Cyclin dependent kinase 4 inhibitor

ABSTRACT

A gene that encodes an inhibitor of CDK4 has been discovered and its genomic nucleotide sequence has been identified. Susceptibility to certain cancers has been shown to be causatively related to the deletion of, or polymorphisms in, the CDK4I gene. The invention is therefore directed to the gene (CDK4I), the inhibitor protein, as well as therapeutic and diagnostic methods which utilize both the CDK4I gene and the CDK4I protein.

This is a continuation of application Ser. No. 08/445,648, filed May 22,1995 (now abandoned), which was a divisional application of Ser. No.08/227,800 filed Apr. 14, 1994.

BACKGROUND OF THE INVENTION

1. Field of the Invention.

The invention relates to the detection of genetic abnormalities thatconfer susceptibility to certain cancers in humans. More specifically,the invention relates to methods for detecting deletions of, orpolymorphisms in, a newly discovered gene which encodes a tumorsuppressor.

2. History of the Prior Art.

In recent years, a growing body of evidence has developed which supportsthe theory that the development of certain tumors is suppressed by geneproducts (“tumor suppressors”) which inhibit cellular proliferation (seee.g., the review in Marx, Science, 263:319-320, 1994). Conversely, ifthe tumor suppressors which would ordinarily be present in a cell areeither absent (due, for example, to a gene deletion) or less active(due, for example, to a gene. mutation), tumor growth which wouldotherwise be inhibited may go unchecked. However, although the growth ofcertain tumors has been positively demonstrated to relate to thedeletion of a tumor suppressor expressing gene, it has not yet beenshown that mutations in the same genes will allow abnormal cellularproliferation to occur.

The growth cycle of eukaryotic cells is regulated by a family of proteinkinases known as the cyclin-dependent kinases (“CDK's”). As shown inFIG. 1, the cyclins and their associated CDK's move cells through thethree phases of the growth cycle (G₁, S and G₂, respectively) leading todivision in the mitosis phase (M). The cyclin/CDK complexes whose rolein cellular proliferation has been most clearly defined to date are thecyclin D/CDK enzymes, which are believed to assist in the progression ofthe G₁ growth cycle phase. Of these enzymes, cyclin D1 is believed to bean oncogene, whose overexpression stimulates excessive cell divisionthrough the continuous production of kinase, thus contributing to thedevelopment of cancers of, for example, the breast and esophagus. CyclinD1 is specifically bound by CDK4 as part of a multiprotein complex thatalso consists of a protein known as p21 and cell nuclear antigen.

Known inhibitors of such cyclin/CDK overexpression include the tumorsuppressor protein p53 and the protein product of the retinoblastoma(Rb) gene. Recently, another putative inhibitor (p16) was isolated and acDNA for the inhibitor was partially sequenced by Serrano, et al,Nature, 366:704-710, 1993. The authors demonstrated that p16 binds CDK4to inhibit the activity of the CDK4/cyclin D enzymes. Based on dataindicating that p16 prevented phosphorylation by CDK/cyclin D of certainRb growth cycle proteins, the authors proposed that p16 acts in vivoupstream and downstream of Rb to form a negative feedback loop toregulate cellular proliferation. However, no connection between p16 andthe occurrence or inhibition of particular cancers was suggested, norhas any information been published concerning the genomic structure ofthe gene encodings p16.

SUMMARY OF THE INVENTION

Prior to the publication of the Serrano, et al., article referred toabove, the inventors discovered a tumor suppressor gene (hereafter,“CDK4I”) and identified its genomic structure (see, SEQ ID NO's: 1-2).In non-malignant cells, CDK4I maps to chromosome 9p21 and is physicallyadjacent to the gene for methylthioadenosine phosphorylase (MTAse) (see,FIG. 4(b)). MTAse deficiencies resulting from deletions of, or mutationsin, the gene for MTAse have been shown to be directly related to theonset of certain cancers (see, Nobori, et al, Cancer Res. 53:1098-1101,1993, the disclosure of which are incorporated herein for referenceregarding the role of MTAse in cancer development, and SEQ ID NO: 14,the nucleotide sequence of genomic MTAse).

Approximately one-half of all tumor cells which have been identified todate as either lacking CDK4I or containing mutations or rearrangements(collectively, “polymorphisms”) of the CDK4I gene also lack MTAse. Theinventors have also identified mutations in the CDK4I gene which arepresent in the tumor cells of patients with certain cancers. Theinvention is therefore directed to methods to detect (a) deletions ofthe CDK4I gene in cells, and (b) polymorphisms, which deletions andpolymorphisms are indicative of susceptibility to certain cancers.

More specifically, in one aspect, the invention comprises methods fordetecting point mutations in, or deletions of, the CDK4I gene. Suchmethods include polymerase chain reaction (PCR) based assays, gelelectrophoresis of single-strand conformation polymorphisms, directsequencing, and restriction endonuclease digestion. Detection of adeletion of the CDK4I gene will preferably be performed by a uniquecompetitive PCR technique.

In another aspect, the invention comprises methods for detection ofCDK4I proteins and biologically active fragments thereof (collectively,“CDK4I”) in a biological cell sample.

In another aspect, the invention comprises screening protocols forsusceptibility to particular cancers based on detection of polymorphismsassociated with the occurrence of the cancers.

In another aspect, the invention comprises screening protocols forsusceptibility to particular cancers based on detection of polymorphismsin, or deletions of, the genes for both CDK4I and MTAse, as well asdetection of deficiencies in the products of the genes.

In another aspect, the invention comprises genomic CDK4I, expressionproducts of the CDK4I gene, CDK4I and fragments thereof, as well asantibodies which will specifically bind CDK4I gene expression products,CDK4I and CDK4I fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the phases from G1 through mitosis (M) of the growthcycle of a mammalian cell.

FIGS. 2 (a-b) depicts the full-length genomic sequence (SEQ ID NOS:1 and2) for the human CDK4I gene, wherein the exons CDK4I5′ (SEQ ID NO:3),CDK4I′ (SEQ ID NO:4) and CDK4I3′ (SEQ ID NO:5) are underlined.

FIG. 3 compares the 5′ regions of the genomic DNA sequence shown in FIG.2 (bottom line; SEQ ID NO:16) with the cDNA sequence reported bySerrano, et al., in Nature, 366:704-710, 1993 (top line; SEQ ID NO:15),wherein differences are indicated by the absence of a vertical linebetween the sequences.

FIGS. 4 (a-b) depicts the region of chromosome 9p21 between the MTAseand INF-α gene loci, focusing on the deleted segment in T98G. FIG. (a)shows the nucleotide sequence of the deleted segment (SEQ ID NOS:17 and18); FIG. (b) shows the relationship of the region to the MTAse andINF-α genes on chromosome 9.

FIG. 5 maps sites of deletions between the 54F and 5BS regions of theregion between the gene loci for MTAse and INF-a, wherein the site ofthe gene for CDK4I is in the most frequently deleted region.

FIG. 6 compares the normal DNA sequence of the CDK4I gene (bottom line;SEQ ID NO:20) and a mutated sequence of the gene (top line; SEQ IDNO:19) containing a single base substitution found in cells from a humanpatient with familial melanoma.

FIG. 7 compares the normal DNA sequence of the CDK4I gene (bottom line;SEQ ID NO:22) and a mutated sequence of the gene (top line; SEQ IDNO:21) containing an intragenic microdeletion found in a leukemia cellline.

FIG. 8 depicts the results of PCR-based assays for the CDK4I gene inseveral human malignant cell lines. Lane1=placental cells, lane2=SK-MEL-31 (ATCC HTB73; a melanoma cell line); lane 3=WM 266-4 (ATCCCRL 1676; a melanoma cell line), lane 4=T98G (a glioma cell line), lane5=BV173, lane 6=CEM (ATCC CCL 119; a lymphoblastic leukemia cell line),lane 7=MOLT-4 (ATCC 1582; a lymphoblastic leukemia), lane 8=A549 (ATCCCCL 185; a non-small cell lung cancer cell line), lane 9=SK-MES-1 (ATCCHTB 58; a non-small cell lung cancer cell line). Lane 10 has notemplates and lane 11 has DNA markers.

FIG. 9 depicts the results of reverse transcriptase PCR-based assays formRNA corresponding to the CDK4I gene in several malignant cell lines.Lane 1=WIL2-NS (ATCC CRL 8155; a normal lymphoblastoid cell line), lane2=U937 (ATCC CRL 1593; a leukemia cell line ), lane 3=T98G (ATCC CRL1690; a glioma cell line), lane 4=H661 (ATCC HTB-183; a non-small celllung cancer cell line), lane 5=A-549 (ATCC CCL 185; a non-small celllung cancer cell line), and lane 6=SK-MES-1 (ATCC HTB 58; a non-smallcell lung cancer cell line). M=DNA markers.

FIG. 10 is the genomic nucleotide sequence for MTAse (SEQ ID NO:23),with the exons underlined.

DETAILED DESCRIPTION OF THE INVENTION I. IDENTIFICATION ANDCHARACTERIZATION OF GENOMIC CDK4I

In the Sequence Listing appended hereto, the full-length genomicnucleotide sequence for the human CDK4I gene (i.e., “CDK4Ipolynucleotide”) is set forth at SEQ ID NO's: 1 and 2 (and is reproducedin FIGS. 2 (a-b)). SEQ ID NO'S 3-5 contain the nucleotide sequences forthe CDK4I gene exons; these exons are underlined in FIGS. 2 (a-b), thusshowing the boundaries between the exons (hereafter, “CDK4I′”, “CDK4I3′”and “CDK4I5′”) as well as introns of the gene. The CDK4I′ exon containsa palindromic region of 4 inverted repeats which likely contribute tothe structural stability of the expressed CDK4I protein. Comparison tothe reported p16 cDNA sequence (Serrano, et al., Nature, supra) revealsthat the reported sequence contains regions encoding for E. coliproteins and differs in its 5′ region from the CDK4I gene by severalbase pairs, (see comparison contained in FIG. 3; the relevant portionsof genomic CDK4I are shown along the bottom line while the Serrano, etal., partial sequence (5′ region) is shown along the top line.Differences in the sequences are indicated by the absence of verticalconnecting lines).

Genomic CDK4I was identified and characterized as described below. TheCDK4I gene was believed to reside on chromosome 9p between the loci forMTAse and the interferon alpha (“INF-a”) gene cluster. This location wassuggested by the fact that many malignant cell lines with deletions inchromosome 9p either lack MTAse or have hemizygous or homozygousdeletions of INF-a. In particular, a small 9p deletion identified in theT98G glioma cell line (ATCC Accession No. CRL 1690) centromeric to theINF-a loci was focused upon as a possible location for CDK4I.

As described in greater detail in Example I, the putative location forCDK4I was explored with a MTAse cDNA that was used to probe a humanplacenta lambda phage library (SEQ ID NO:14 contains the genomicnucleotide sequence for MTAse; see also, ATCC Accession Nos.55536-55540). Starting with a 2 kilobase Hind III fragment (MTAse clone7-2; ATCC Accession No. 55540), chromosome walking was performed and,through screening of subsequent lambda phage libraries, clones wereisolated which encompassed the deleted region in T98G cells. The regionof chromosome 9p21 between the loci for the MTAse gene and the INF-αgene was sequenced focusing on the deleted segment in T98G; the sequenceis contained in FIG. 4(a) (SEQ ID NOS:17 and 18).

45 cancer cell lines were screened to determine the frequency ofdeletions of the putative tumor suppressor gene and other sites inregion identified in FIG. 4(a). Data obtained from this assay are shownin FIG. 8. Introns from the two most frequently deleted sites areidentified in FIG. 4(b) as sequence tagged site (STS) 54F and STS 5BS,which sites are separated by 50 kilobase region. Probes were designed tospecifically bind to portions of the 50 kilobase region between STS 54Fand STS 5BS (SEQ ID NOS:6 and 7). The most frequently deleted region wasidentified by a 19 kilobase lambda phage clone (10B1-10) (see, FIG.4(a); SEQ ID NOS:17 and 18). As described in Example I, the CDK4I genewas found to reside in the region of chromosome 9 which corresponds toclone 10B1-10 (CDK4I3′ and CDK4I′) and a related clone 10A1 (CDK4I5′).

The CDK4I gene is contained in two E. coli strains (containing,respectively, 10B1-10 and 10A1) on deposit with the American TypeCulture Collection (“ATCC”), at P.O. Box 1549, Manassas, Va. 20108deposited on Apr. 14, 1994 under Designation Nos. 69606 and 69607(respectively, clone 10B1-10 [SEQ ID NO: 1] and 10A-10 [SEO ID NO: 2]).However, no admission that this deposit was necessary to the enablementof this disclosure or any of the claims contained herein is made orintended.

As shown in FIG. 2 and SEQ ID NO's: 3-5, the CDK4I exon of the CDK4Igene has a 306 base pair open reading frame, the CDK4I3′ exon has ashort open reading frame corresponding to the last 15 base pairs of thecoding region for CDK4I and the CDK4I5′ exon has a 139 base pair openreading frame.

II. FREQUENCY OF DELETION OF THE CDK4I GENE IN CANCER CELL LINES

Many cancers cluster in families. For example, of approximately 30,000new cases of cutaneous melanomas diagnosed annually in the UnitedStates, about 5-10% originate in a familial setting (see,Cannon-Albright, et al., Science, 258:1148-1152). The locus for familialmelanoma has previously been identified as chromosome 9p21, a regionthat is reproducibly deleted in sporadic melanomas (Fountain, et aL,Proc.Natl.Acad.Sci.USA, 89:10557-10561, 1992). In addition,environmental factors, such as exposure to ultraviolet rays andcigarette smoking have been identified as major risk factors for thedevelopment of melanomas in the former case and of lung, bladder, head,neck, and larynx cancers. For example, abnormalities of chromosome 9p21are very common in lung cancer cells (Nobori, et al., Cancer Res.,53:1098-1101, 1993).

As described in Example II, to determine whether the CDK4I gene waspresent in, or deleted from, known cancer cell lines, probescorresponding to the CDK4I gene were used to rescreen the 45 cancer celllines referred to above. The results of this assay are shown (in ahybridization blot) in FIG. 9. For reference, probes corresponding tothe MTAse, INF-a and INF-b genes, as well as the 3.21, 2F, 54F, 71F, and3.3B regions on chromosome 9 (see, FIG. 4 (b) and FIG. 5) were used toscreen for the presence of those regions in the same cell lines. Thecomplete results of this assay for all gene regions tested are tabulatedby percentage deletion in Table 1 below, to wit; 61% of melanomas, 87%of gliomas, 36% of non-small lung cancers and 64% of leukemias wereidentified as having homozygous deletions of the CDK4I gene. These dataindicate that human cells contain a single CDK4I gene that is deleted orrearranged in the majority of melanomas, gliomas, and leukemias, as wellas more than a third of non-small cell lung cancers.

TABLE 1 HOMOZYGOUS LOSS OF CHROMOSOME 9p LOCI IN HUMAN CANCER CELL LINESCell Line (Number tested) MTAP 3.21 2F 54F CDK4I 5BS 71F 3.3B IFNA8 IFNBMelanoma (13) 30.8 38.5 53.8 53.8 61.5 61.5 61.5 15.4 7.7 0 Glioma (8)62.5 75.0 87.5 87.5 87.5 75.0 75.0 62.5 62.5 25.0 Lung Cancer (11) 27.327.3 27.3 27.3 45.5 45.5 45.5 9.1 9.1 0 Leukemia (14) 50.0 50.0 64.364.3 64.3 57.1 57.1 28.6 28.6 21.4

III. FREQUENCY AND IDENTITY OF POINT MUTATIONS OF THE CDK4I GENE INTUMOR CELLS

As discussed in the background section above, the gene encoding thetumor suppressor p53 has been found to be deleted in certain cancers,thus allowing unchecked cellular proliferation to occur. Logically, if agene encoding a tumor suppressor contains a polymorphism thatcompromises the activity of the suppressor, then tumors may develop overtime even without deletion of the gene encoding the suppressor. In theparticular case of the CDK4I gene, its presence on chromosome 9p21suggests that both deletions and polymorphisms of the gene maycontribute to the onset of certain familial and environmental cancers.

More specifically, the role of CDK4I in binding and inhibiting CDK4indicates that an excessive level of kinases can be expected to developwithin cells that harbor a CDK4I gene deletion or polymorphism thatcompromises the ability of CDK4I to inhibit CDK4. Thus, while deletionsof the CDK4I gene will be indicative of a pre-malignancy or malignancy,polymorphisms in the gene (particularly polymorphisms in germline cellsof persons with a familial history of 9p21-linked cancers) will beindicative of a susceptibility to develop a “cancer condition” (i.e., acondition which is causatively related to excessive cellular levels ofCDK4).

In its broadest sense, the present invention allows the detection of anypolymorphism in, or deletion of, a CDK4I target nucleic acid sequence ofdiagnostic or therapeutic relevance, where the target nucleic acidsequence is present in a biological cell sample such as that heretoforesubjected to histopathologic examination using techniques of lightmicroscopy, such as the margins of a primary tumor or a regional lymphnode. Thus, the target nucleotide sequence may be, for example, a mutantnucleotide, a restriction fragment length polymorphism (RFLP), anucleotide deletion, a nucleotide substitution, or any other mammaliannucleic acid sequence of interest in such tissue specimens. As usedherein the term “polymorphism” as applied to a target CDK4I nucleotidesequence shall be understood to encompass a mutation, a restrictionfragment length polymorphism, a nucleic acid deletion, or a nucleic acidsubstitution.

For example, cells from a human patient who had been diagnosed assuffering from familial melanoma (specifically, dysplastic nevussyndrome) were identified as containing a nonsense mutation (i.e., a Cto T transition) at position 166 of the CDK4I mRNA (see, FIG. 6 (SEQ IDNOS:19 and 20) and Example V). In addition, cells from a known leukemiacell line (U937; ATCC Accession No. 1593) were screened and found tocontain an intragenic microdeletion of 18 base pairs in the CDK4I5′ exon(see, FIG. 7 (SEQ ID NOS:21 and 22) and Example VI). Using theinformation contained in SEQ ID NOS:1 and 2 and techniques foridentifying point mutations in genes which are well-known in the art andillustrated herein, those of ordinary skill in the art will be able toscreen cell samples from particular 9p21-linked tumors for reproduciblepolymorphisms and/or deletions of CDK4I to determine geneticsusceptibility to, as well as the existence of, a cancer condition asdefined herein (particularly melanomas, gliomas, non-small cell lungcancers and leukemias).

In the case of deletions and polymorphisms, this information can be usedto diagnose a pre-cancerous condition or existing cancer condition.Further, by quantitating the number of cells in successive cell sampleswhich bear and acquire the deletion or polymorphism at separatelocations in the body and/or over time, the progression of a cancercondition can be monitored. Similarly, where a deletion or polymorphismis found in a patient who has not yet developed symptoms of a cancercondition (particularly one who carries the abnormality in germlinecells and/or has a family history of a particular cancer condition), thedeletion or polymorphism will be indicative of a genetic susceptibilityto develop the cancer condition. Such susceptibility can further beevaluated on a qualitative basis based on information concerning theprevalence, if any, of the cancer condition in the patients familyhistory and the presence of other risk factors, such as exposure toenvironmental factors and whether the patient also carries cells havinga deletion of the gene for MTAse.

To this end, preferred diagnostic techniques are described below, theuse of which is illustrated in the Examples provided herein.

IV. METHODS FOR DETECTION OF DELETIONS AND POLYMORPHISMS IN THE CDK4IGENE

Amplification of the CDK4I gene is generally required to producedetectable amounts of any gene present in a biological cell sample;i.e., a fluid or tissue sample which includes a sample of germline cells(e.g., from blood, skin or hair follicles) or somatic cells in amalignant or pre-malignant lesion (e.g., from tissue biopsies, sputum orurinary specimens). Following amplification, point mutations may bedetected by means known to those of ordinary skill in the art such asdirect sequencing, or oligonucleotide hybridization under conditionsthat can detect a single base pair change. Also suitable are thetechniques for gel electrophoresis of single strand conformationpolymorphisms (known in the art as “SSCP”; see, e.g., Orita, et al.,Proc.Natl.Acad,Sci.USA, 86:2766-2770,1989),heteroduplex analysis todetect mismatches between double stranded DNA (a suitable kit for thisprotocol is the “MDE Heteroduplex Kit” sold by AT Biochem of Malvern,Pa.), allele specific PCR (see e.g., Wu, et al, Proc.Natl.Acad.Sci.USA,86:2757-2760, and restriction fragment length polymorphism analysis(known in the art as “RFLP”; see, e.g., Knowlton, et al., Nature,318:380-382, 1985). Examples of the application of these techniques todetect polymorphisms in the CDK4I gene are provided infra; for furtherdetails, the disclosures of the references referred to in the precedingsentence are incorporated herein by this reference.

Detection of homozygous deletions of the CDK4I gene may be readilydetected by known PCR techniques, as illustrated further below. However,it is possible for a person to be hemizygous for the CDK4I gene, inwhich case gene dosage analysis for each exon will be performed.Quantitative PCR techniques known in the art may be used to perform thisanalysis; a preferred technique is described below and in Kohsaka, etal, Nuc.Acids Res., 21:3469-3472, 1993. Examples illustrating the use ofthe preferred technique to detect point mutations in the CDK4I gene areprovided infra; for further reference, the disclosures of the Kohsaka,et al., article and co-pending applications referred to in the precedingsentence are incorporated herein by reference.

The most preferred method for performance of qualtitative PCR to detectdeletions and polymorphisms of the CDK4I gene involves use of thePCR-ELISA techniques described in infra and in Kohsaka, et al., supra.Although such PCR-ELISA methods are preferred for their sensitivity andsimplicity, those of ordinary skill in the art will know of, or canreadily ascertain, other suitable PCR assays (such as are described in“PCR Protocols”, Innis, et al., eds., (Academic Press, 1990)).

A. General Methods for Use in PCR and PCR-based Assays

When is is desirable to amplify the CDK4I target nucleotide sequencebefore detection, such as a CDK4I nucleotide sequence containing apolymorphism, this can be accomplished using oligonucleotide(s) that areprimers for amplification. These unique oligonucleotide primers arebased upon identification of the lanking regions contiguous with theCDK4I nucleotide sequence containing the polymorphism.

In general, primers for use in PCR-based assays will embraceoligonucleotides of sufficient length and appropriate sequence whichprovides specific initiation of polymerization of a significant numberof nucleic acid molecules containing the target nucleic acid under theconditions of stringency for the reaction utilizing the primers. In thismanner, it is possible to selectively amplify the specific targetnucleic acid sequence containing the nucleic acid of interest.Specifically, the term “primer” as used herein refers to a sequencecomprising two or more deoxyribonucleotides or ribonucleotides,preferably at least eight, which sequence is capable of initiatingsynthesis of a primer extension product that is substantiallycomplementary to a target nucleic acid strand. The oligonucleotideprimer typically contains 15-22 or more nucleotides, although it maycontain fewer nucleotides as long as the primer is of sufficientspecificity to allow essentially only the amplification of thespecifically desired target nucleotide sequence (i.e., the primer issubstantially complementary).

Experimental conditions conducive to synthesis include the presence ofnucleoside triphosphates and an agent for polymerization, such as DNApolymerase, and a suitable temperature and pH. The primer is preferablysingle stranded for maximum efficiency in amplification, but may bedouble stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent for polymerization. The exact length ofprimer will depend on many factors, including temperature, buffer, andnucleotide composition.

Primers for use in the PCR-based assays of the invention will bedesigned to be “substantially” complementary to each strand of mutantnucleotide sequence to be amplified. Substantially complementary meansthat the primers must be sufficiently complementary to hybridize withtheir respective strands under conditions which allow the agent forpolymerization to function. In other words, the primers should havesufficient complementarily with the flanking sequences to hybridizetherewith and permit amplification of the mutant nucleotide sequence.Preferably, the 3′ terminus of the primer that is extended has perfectlybase paired complementarity with the complementary flanking strand.

Oligonucleotide primers used according to the invention are employed inany amplification process that produces increased quantities of targetnucleic acid. Typically, one primer is complementary to the negative (−)strand of the mutant nucleotide sequence and the other is complementaryto the positive (+) strand. Annealing the primers to denatured nucleicacid followed by extension with an enzyme, such as the large fragment ofDNA Polymerase I (Kienow) or Taq DNA polymerase and nucleotides orligases, results in newly synthesized + and − strands containing thetarget nucleic acid. Because these newly synthesized nucleic acids arealso templates, repeated cycles of denaturing, primer annealing, andextension results in exponential production of the region (i.e., thetarget mutant nucleotide sequence) defined by the primer. The product ofthe amplification reaction is a discrete nucleic acid duplex withtermini corresponding to the ends of the specific primers employed.Those of skill in the art will know of other amplification methodologieswhich can also be utilized to increase the copy number of target nucleicacid.

The oligonucleotide primers for use in the invention may be preparedusing any suitable method, such as conventional phosphotriester andphosphodiester methods or automated embodiments thereof. In one suchautomated embodiment, diethylphosphoramidites are used gas startingmaterials and may be synthesized as described by Beaucage, et al.(Tetrahedron Letters, 22:1859-1862, 1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. One method of amplification which can be used accordingto this invention is the polymerase chain reaction (PCR) described inU.S. Pat. Nos. 4,683,202 and 4,683,195.

The nucleic acid from any biological cell sample, in purified ornonpurified form, can be utilized as the starting nucleic acid or acids,provided it contains, or is suspected of containing, the specificnucleic acid sequence containing the target nucleic acid. Thus, theprocess may employ, for example, DNA or RNA, including messenger RNA(mRNA), wherein DNA or RNA may be single stranded or double stranded. Inthe event that RNA is to be used as a template, enzymes, and/orconditions optimal for reverse transcribing the template to DNA would beutilized. In addition, a DNA-RNA hybrid which contains one strand ofeach may be utilized. A mixture of nucleic acids may also be employed,or the nucleic acids produced in a previous amplification reactionherein, using the same or different primers may be so utilized. Themutant nucleotide sequence to be amplified may be a fraction of a largermolecule or can be present initially as a discrete molecule, such thatthe specific sequence constitutes the entire nucleic acid. It is notnecessary that the sequence to be amplified be present initially in apure form; it may be a minor fraction of a complex mixture, such ascontained in whole human DNA.

Where the target neoplastic nucleotide sequence of the sample containstwo strands, it is necessary to separate the strands of the nucleic acidbefore it can be used as the template. Strand separation can be effectedeither as a separate step or simultaneously with the synthesis of theprimer extension products. This strand separation can be accomplishedusing various suitable denaturing conditions, including physical,chemical, or enzymatic means; the word “denaturing” includes all suchmeans. One physical method of separating nucleic acid strands involvesheating the nucleic acid until it is denatured. Typical heatdenaturation may involve temperatures ranging from about 80° to 105° C.for times ranging from about 1 to 10 minutes. Strand separation may alsobe induced by an enzyme from the class of enzymes known as helicases orby the enzyme RecA, which has helicase activity, and in the presence ofriboATP which is known to denature DNA. The reaction conditions suitablefor strand separation of nucleic acids with helicases are described byKuhn Hoffmann-Berling (CSH-Quantitative Biology, 43:63, 1978) andtechniques for using RecA are reviewed in C. Radding (Ann. Rev.Genetics, 16:405-437, 1982).

If the nucleic acid containing the target nucleic acid to be amplifiedis single stranded, its complement is synthesized by adding one or twooligonucleotide primers. If a single primer is utilized, a primerextension product is synthesized in the presence of primer, an agent forpolymerization, and the four nucleoside triphosphates described below.The product will be complementary to the single-stranded nucleic acidand will hybridize with a single-stranded nucleic acid to form a duplexof unequal length strands that may then be separated into single strandsto produce two single separated complementary strands. Alternatively,two primers may be added to the single-stranded nucleic acid and thereaction carried out as described.

When complementary strands of nucleic acid or acids are separated,regardless of whether the nucleic acid was originally double or singlestranded, the separated strands are ready to be used as a template forthe synthesis of additional nucleic acid strands. This synthesis isperformed under conditions allowing hybridization of primers totemplates. Generally synthesis occurs in a buffered aqueous solution,preferably at a pH of 7-9, most preferably about 8. Preferably, a molarexcess (for genomic nucleic acid, usually about 10⁸:1 primer:template)of the two oligonucleotide primers is added to the buffer containing theseparated template strands. It is understood, however, that the amountof complementary strand may not be known if the process of the inventionis used for diagnostic applications, so that the amount of primerrelative to the amount of complementary strand cannot be determined withcertainty. As a practical matter, however, the amount of primer addedwill generally be in molar excess over the amount of complementarystrand (template) when the sequence to be amplified is contained in amixture of complicated long-chain nucleic acid strands. A large molarexcess is preferred to improve the efficiency of the process.

In some amplification embodiments, the substrates, for example, thedeoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP, are addedto the synthesis mixture, either separately or together with theprimers, in adequate amounts and the resulting solution is heated toabout 90°-100° C. from about 1 to 10 minutes, preferably from 1 to 4minutes. After this heating period, the solution is allowed to cool toroom temperature, which is preferable for the primer hybridization. Tothe cooled mixture is added an appropriate agent for effecting theprimer extension reaction (called herein “agent for polymerization”),and the reaction is allowed to occur under conditions known in the art.The agent for polymerization may also be added together with the otherreagents if it is heat stable. This synthesis (or amplification)reaction may occur at room temperature up to a temperature above whichthe agent for polymerization no longer functions. Thus, for example, ifDNA polymerase is used as the agent, the temperature is generally nogreater than about 40° C.

The agent for polymerization may be any compound or system which willfunction to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose include, forexample, E. coli DNA polymerase I, Taq polymerase, KIenow fragment of E.coli DNA polymerase I, T4 DNA polymerase, other available DNApolymerases, polymerase muteins, reverse transcriptase, ligase, andother enzymes, including heat-stable enzymes (i.e., those enzymes whichperform primer extension after being subjected to temperaturessufficiently elevated to cause denaturation). Suitable enzymes willfacilitate combination of the nucleotides in the proper manner to formthe primer extension products which are complementary to each mutantnucleotide strand. Generally, the synthesis will be initiated at the 3′end of each primer and proceed in the 5′ direction along the templatestrand, until synthesis terminates, producing molecules of differentlengths. There may be agents for polymerization, however, which initiatesynthesis at the 5′ end and proceed in the other direction, using thesame process as described above. In any event, the method of theinvention is not to be limited to the embodiments of amplification whichare described herein.

The newly synthesized mutant nucleotide strand and its complementarynucleic acid strand will form a double-stranded molecule underhybridizing conditions described above and this hybrid is used insubsequent steps of the process. In the next step, the newly synthesizeddouble-stranded molecule is subjected to denaturing conditions using anyof the procedures described above to provide single-stranded molecules.

The above process is repeated on the single-stranded molecules.Additional agent for polymerization, nucleosides, and primers may beadded, if necessary, for the reaction to proceed under the conditionsprescribed above. Again, the synthesis will be initiated at one end ofeach of the oligonucleotide primers and will proceed along the singlestrands of the template to produce additional nucleic acid. After thisstep, half of the extension product will consist of the specific nucleicacid sequence bounded by the two primers.

The steps of denaturing and extension product synthesis can be repeatedas often as needed to amplify the target mutant nucleotide sequence tothe extent necessary for detection. The amount of the mutant nucleotidesequence produced will accumulate in an exponential fashion.

The amplified product may be detected by Southern blot analysis, withoutusing radioactive probes. In such a process, for example, a small sampleof DNA containing a very low level of mutant nucleotide sequence isamplified, and analyzed via a Southern blotting technique. The use ofnon-radioactive probes or labels is facilitated by the high level of theamplified signal.

Nucleic acids having a mutation detected in the method of the inventioncan be further evaluated, detected, cloned, sequenced, and the like,either in solution or after binding to a solid support, by any methodusually applied to the detection of a specific DNA sequence such as PCR,oligomer restriction (Saiki, et al., Bio/Technology, 3:1008-1012, 1985),allele-specific oligonucleotide (ASO) probe analysis (Conner, et al.,Proc. Natl. Acad. Sci. USA, 80:278, 1983), oligonucleotide ligationassays (OLAs) (Landegren, et al., Science, 241:1077, 1988), and thelike. Molecular techniques for DNA analysis have been reviewed(Landegren, et al., Science, 242:229-237, 1988).

B. Hybridization with Labelled Probes

In another diagnostic method of the invention, purified nucleic acidfragments containing intervening sequences oroligonucleotide sequencesof 10-50 base pairs are radioactively labelled. The labelledpreparations are used to probe nucleic acid from a biological cellsample by the Southern hybridization technique. Nucleotide fragmentsfrom a biological cell sample, before or after amplification, areseparated into fragments of different molecular masses by gelelectrophoresis and transferred to filters that bind nucleic acid. Afterexposure to the labelled probe, which will hybridize to nucleotidefragments containing target nucleic acid sequences, binding of theradioactive probe to target nucleic acid fragments is identified byautoradiography (see Genetic Engineering, 1, ed. Robert Williamson,Academic Press, (1981), 72-81). Alternatively, nucleic acid from thesample can be bound directly to filters to which the radioactive probeselectively attaches by binding nucleic acids having the sequence ofinterest. Specific sequences and the degree of binding is quantitated bydirectly counting the radioactive emissions.

Where the target nucleic acid is not amplified, detection using anappropriate hybridization probe may be performed directly on theseparated mammalian nucleic acid. In those instances where the targetnucleic acid is amplified, detection with the appropriate hybridizationprobe would be performed after amplification.

The probes of the present invention can be used for examining thedistribution of the specific fragments detected, as well as thequantitative (relative) degree of binding of the probe for determiningthe occurrence of specific strongly binding (hybridizing) sequences,thus indicating the likelihood for an individual to be at low risk orhigh risk for a cancer condition, such as familial melanoma.

For the most part, the probe will be detectably labelled with an atom orinorganic radical, most commonly using radionuclides, but also heavymetals can be used. Conveniently, a radioactive label may be employed.Radioactive labels include ³²p, ¹²⁵I, ³H, ¹⁴C, ¹¹¹In, ^(99m)Tc, or thelike. Any radioactive label may be employed which provides for anadequate signal and has sufficient half-life. Other labels indudeligands, which can serve as a specific binding pair member for alabelled ligand, and the like. A wide variety of labels routinelyemployed in immunoassays can readily be employed in the present assay.

The choice of the label will be governed by the effect of the label onthe rate of hybridization and binding of the probe to the targetnucleotide sequence. It will be necessary that the label providesufficient sensitivity to detect the amount of target nucleotidesequence available for hybridization. Other considerations will be easeof synthesis of the probe, availability of instrumentation, ability toautomate, convenience, and the like.

The manner in which the label is bound to the probe will vary dependingupon the nature of the label. For a radioactive label, a wide variety oftechniques can be employed. Commonly employed is nick translation withan a ³²P-dNTP or terminal phosphate hydrolysis with alkaline phosphatasefollowed by labeling with radioactive ³²P employing ³²P-NTP and T4polynucleotide kinase. Altematively, nucleotides can be synthesizedwhere one or more of the elements present are replaced with aradioactive isotope, e.g., hydrogen with tritium. If desired,complementary labelled strands can be used as probes to enhance theconcentration of hybridized label.

Where other radionucleotide labels are involved, various linking groupscan be employed. Aterminal hydroxyl can be esterified, with inorganicacids, e.g., ³²P phosphate, or ¹⁴C organic acids, or else esterified toprovide linking groups to the label. Alternatively, intermediate basesmay be substituted with activatable linking groups that can then belinked to a label.

Enzymes of interest as reporter groups will primarily be hydrolases,particularly esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,and so forth. Chemiluminescers include luciferin, and 2,3-dihydrophthalazinediones (e.g., luminol).

The probe can be employed for hybridizing to a nucleotide sequenceaffixed to a water insoluble porous support. Depending upon the sourceof the nucleic acid, the manner in which the nucleic acid is affixed tothe support may vary. Those of ordinary skill in the art know, or caneasily ascertain, different supports that can be used in the method ofthe invention.

The nucleic acid from a biological cell sample is cloned and thenspotted or spread onto a filter to provide a plurality of individualportions (plaques). The filter is an inert porous solid support, e.g.,nitrocellulose. Any cells (or phage) present in the specimen are treatedto liberate their nucleic acid. The lysing and denaturation of nucleicacid, as well as the subsequent washings, can be achieved with anappropriate solution for a sufficient time to lyse the cells anddenature the nucleic acid. For lysing, chemical lysing will convenientlybe employed, as described previously for the lysis buffer. Otherdenaturation agents include elevated temperatures, organic reagents,e.g., alcohols, amides, amines, ureas, phenols and sulfoxides or certaininorganic ions, e.g., thiocyanate and perchlorate.

After denaturation, the filter is washed in an aqueous bufferedsolution, such as Tris, generally at a pH of about 6 to 8, usually 7.One or more washings may be involved, conveniently using the sameprocedure as employed for the lysing and denaturation. After the lysing,denaturing, and washes have been accomplished, the nucleic acid spottedfilter is dried at an elevated temperature, generally from about 50° C.to 70° C. Under this procedure, the nucleic acid is fixed in positionand can be assayed with the probe when convenient.

Pre-hybridization may be accomplished by incubating the filter with thehybridization solution without the probe at a mildly elevatedtemperature for a sufficient time to thoroughly wet the filter. Varioushybridization solutions may be employed, comprising from about 20% to60% volume, preferably 30%, of an inert polar organic solvent. A commonhybridization solution employs about 50% formamide, about 0.5 to 1Msodium chloride, about 0.05 to 0.1M sodium citrate, about 0.05 to 0.2%sodium dodecylsulfate, and minor amounts of EDTA, ficoll (about 300-500kD), polyvinylpyrrolidone, (about 250-500 kD) and serum albumin. Alsoincluded in the hybridization solution will generally be from about 0.5to 5 mg/ml of sonicated denatured DNA, e.g., calf thymus of salmonsperm; and optionally from about 0.5 to 2% wt/vol glycine. Otheradditives may also be included, such as dextran sulfate of from about100 to 1,000 kD and in an amount of from about 8 to 15 weight percent ofthe hybridization solution.

The particular hybridization technique is not essential to theinvention. Other hybridization techniques are described by Gall andPardue, (Proc. Nat. Acad. Sci. 63:378, 1969); and John, et al., (Nature,223:582, 1969). As improvements are made in hybridization techniquesthey can readily be applied in the method of the invention.

The amount of labelled probe present in the hybridization solution willvary widely, depending upon the nature of the label, the amount of thelabelled probe that can reasonably bind to the filter, and thestringency of the hybridization. Generally, substantial excess overstoichiometric concentrations of the probe will be employed to enhancethe rate of binding of the probe to the fixed target nucleic acid.

Various degrees of stringency of hybridization may be employed. The moresevere the conditions, the greater the complementarily that is requiredfor hybridization between the probe and the single stranded targetnucleic acid sequence for duplex formation. Severity can be controlledby temperature, probe concentration, probe length, ionic strength, time,and the like. Conveniently, the stringency of hybridization is varied bychanging the polarity of the reactant solution by manipulating theconcentration of formamide in the range of 20% to 50%. Temperaturesemployed will normally be in the range of about 20° C. to 80° C.,usually 30° C. to 75° C. (see, generally, Current Protocols in MolecularBiology, Ausubel, ed., Wiley & Sons, 1989).

After the filter has been contacted with a hybridization solution at amoderate temperature for a period of time sufficient to allowhybridization to occur, the filter is then introduced into a secondsolution having analogous concentrations of sodium chloride, sodiumcitrate and sodium dodecylsulfate as provided in the hybridizationsolution. The time the filter is maintained in the second solution mayvary from five minutes to three hours or more. The second solutiondetermines the stringency, dissolving cross duplexes and shortcomplementary sequences. After rinsing the filter at room temperaturewith dilute sodium citrate-sodium chloride solution, the filter may nowbe assayed for the presence of duplexes in accordance with the nature ofthe label. Where the label is radioactive, the filter is dried andexposed to X-ray film.

The label may also comprise a fluorescent moiety that can then be probedwith a specific antifluorescent antibody. For example, horseradishperoxidase enzyme can be conjugated to this antibody to catalyze achemiluminescent reaction. Production of light can then be seen on rapidexposure to film.

C. Preferred, Competitive PCR-based Assays

The preferred method for performance of quantitative PCR in theinvention is a competitive PCR technique performed using a competitortemplate containing an induced mutation of one or more base pairs whichresults in the competitor differing in sequence (but not size) from thetarget CDK4I gene template. One of the primers is biotinylated or,preferably, aminated so that one strand (usually the antisense strand)of the resulting PCR product can be immobilized via an amino-carboxyl,amino-amino, biotin-streptavidin or other suitably tight bond to a solidphase support which has been tightly bound to an appropriate reactant.Most preferably, the bonds between the PCR product, solid phase supportand reactant will be covalent ones, thus reliably rendering the bondsresistant to uncoupling under denaturing conditions.

Once the aminated or biotinylated strands of the PCR products areimmobilized, the unbound complementary strands are separated in analkaline denaturing wash and removed from the reaction environment.Sequence-specific oligonucleotides (“SSO's”) corresponding to the targetand competitor nucleic acids are labelled with a detection tag. TheSSO's are then hybridized to the antisense strands in absence ofcompetition from the removed unbound sense strands. Appropriate assayreagents are added and the degree of hybridization is measured by ELISAmeasurement means appropriate to the detection tag and solid phasesupport means used, preferably an ELISA microplate reader. The measuredvalues are compared to derive target nucleic acid content, using astandard curve separately derived from PCR reactions amplifyingtemplates including target and competitor templates.

This method is advantageous in that it is quantitative, does not dependupon the number of PCR cycles, and is not influenced by competitionbetween the SSO probe and the complementary strand in the PCR product.

Alternatively, part of the polymerization step and all of thehybridization step can be performed on a solid phase support. In thismethod, it is an nucleotide polymerization primer (preferably anoligonucleotide) which is captured onto a solid phase support ratherthan a strand of the PCR products. Target and competitor nucleic acidPCR products are then added in solution to the solid phase support and apolymerization step is performed. The unbound sense strands of thepolymerization product are removed under the denaturing conditionsdescribed above.

A target to competitor nucleic acid ratio can be determined by detectionof labelled oligonucleotide SSO probes using appropriate measurementmeans (preferably ELISA readers) and standard curve as described supra.The efficiency of this method can be so great that a chain reaction inthe polymerization step may be unnecessary, thus shortening the timeneeded to perform the method. The accuracy of the method is alsoenhanced because the final polymerization products do not have to betransferred from a reaction tube to a solid phase support forhybridization, thus limiting the potential for their loss or damage. Ifnecessary for a particular sample, however, the PCR may be used toamplify the target and competitor nucleic acids in a separate reactiontube, followed by a final polymerization performed on the solid phasesupport.

An additional alternative to the above described techniques performs thepolymerization step in a single step on a solid phase support. In thismethod, the PCR is performed to amplify the target (and where aquantitative analysis is desired, the competitor) nucleic acid on asolid phase support. Before the PCR is performed, primers (whichcorrespond to the target and competitor nucleic acids) are tightly boundto the solid phase support. Two additional primers are placed intosolution with the target nucleic acid (or three primers where acompetitive template is present).

As the PCR begins, the templates do not interact with the bound primerto a substantial degree because template concentration is relatively lowand the bound primer is not readily accessible. However, as thetemplates are amplified, more of the PCR products become bound to thesolid phase via hybridization with the bound primer. In essence,therefore, the bound primers serve as hybridization probes for the PCRproducts formed by priming of the target and competitor nucleic acids.Once hybridization occurs, the hybridization primer elongates via thePCR.

Molecules capable of providing different, detectible signals indicativeof the formation of bound PCR products known to those skilled in the art(such as the labels described supra as well as labelled nucleotidechromophores which will form different colors indicative of theformation of target and competitor PCR products) can be added to thereaction solution during the last few cycles of the reaction. The ratiobetween the target and competitor nucleic acids can also be determinedby ELISA or other appropriate measurement means and reagents reactivewith detection tags coupled to the 3′ end of the immobilizedhybridization primers. This method may also be adapted to detect whethera particular gene is present in the sample (without quantifying it) byperforming a conventional noncompetitive PCR protocol.

Those of ordinary skill in the art will know, or may readily ascertain,how to select suitable primers for use in the above methods. Forexample, primers which will amplify the CDK4I gene and correspond to theCDK4I′, CDK413′ and CDK415′ exons are described in SEQ.ID.Nos.8-13.

D. Single-strand Conformation Polymorphism Analysis

Techniques to detect DNA polymorphisms based on restriction fragmentlength polymorphism analysis (RFLP) and electrophoresis gel mobilityshifts caused by single nucleotide substitution in single-stranded DNA(SSCP) have proved to be useful methods for distinguishing allelicvariations at chromosomal loci. For example, RFLP has been used todetect genetic abnormalities present in cystic fibrosis and otherhereditary disorders (see, e.g., Knowlton, et al., Nature, 318:380-382[re use of RFLP to detect cystic fibrosis], and Shiraishi, et al.,Jpn.J.Cancer Res., 78:1302-1308, 1987 [re. performance of RFLPgenerally], the disclosures of which are incorporated herein by thisreference to illustrate knowledge in the art concerning the use ofRFLP). However, RFLP requires that the polymorphisms of interest bepresent in the recognition sequences for the corresponding restrictionendonucleases or when deletion or insertion of a short sequence ispresent in the region detected by a particular probe. Therefore, SSCP isa preferred technique for detection of allele-specific polymorphisms.

The technique for performance of SSCP is well-known in the art (see,e.g., Orita, et at, Genetics, 86:2766-2770, 1989, the disclosure ofwhich is incorporated herein by this reference to illustrate knowledgein the art concerning the use of SSCP). Generally, gene fragments oralleles of interest are denatured and subjected to electrophoresis in aneutral polyacrylamide gel. Single-stranded DNA's (or RNA copiesthereof) are transferred to a membrane (by blotting) and hybridized withdetectably labelled DNA probes for the fragments/alleles of interest.The relative speed in which the fragments/alleles of interest move inthe gel (“mobility shift”) is indicative of the presence or absence ofbase substitutions.

A particularly suitable SSCP technique is one-which uses the PCR is usedto simultaneously amplify the target sequence and label it with aradioisotope or, preferably, a fluorescein molecule (using labelledprimers in the PCR; i.e., “F-PCR-SSCP”). Most preferably, detection ofbands of DNA in a polyacrylamide gel will be performed with an automaticDNA sequencer, which permits strict control of the gel at any desiredtemperature and allows for quantitative interpretation of the resultingdata (based on the proportionality of the heights of the peaks in thefluorogram to the intensity of the fluorescence emitted by the labelledDNA). For a summary of the known method for performance of F-PCR-SSCP,those of skill in the art may wish to consult Makino, et at, PCR Methodsand Appins., 2:10-13 (Cold Spring Harbor Lab., 1992), the disclosure ofwhich is incorporated herein by this reference to illustrate knowledgein the art concerning F-PCR-SSCP.

cl E. Allele-specific Enzvmatic Amplification of Genomic DNA

A simple, and therefore preferred, method of detecting polymorphisms ingenomic DNA is a technique which is based on a allele-specific PCR(ASPCR). In ASPCR, two allele-specific oligonucleotide primers (such asthose described in SEQ ID NO's: 8-13), one of which is specific for thesuspected and/or known mutated allele, the other of which is specificfor the “normal” allele, are used in the PCR with genomic DNA templatesand another primer which is complementary to both alleles. Under theproper annealing temperature and PCR conditions, the primers will onlydirect amplification of their complementary allele, thus allowing forthe determination of genotypes in nucleic acid samples obtained fromhuman tissue. More particularly, suitable temperatures for this PCR areabout 55° C. for the annealing cycles, about 72° C. for thepolymerization cycles, and about 94° C. for the heat-denaturationcycles.

For further details concerning performance of the ASPCR, those of skillin the art may wish to consult Wu, et al., Proc.Natl.Acad.Sci. USA,86:2757-2760, 1989, the disclosure of which is incorporated herein bythis reference.

F. Indirect Detection of Gene Deletions Based on the Absence of CDK4I Ina Biological Cell Sample

In a normal, non-malignant cell, CDK4I can be expected to be present,usually in bound form; i.e., in a complex of CDK4I, CDK4, cyclin D andother molecules, such as a cell nuclear antigen. Methods for indirectdetection of a deletion of the gene for CDK4I based on the absence ofthe CDK4 protein (as determined by, preferably, immunoassay) aredescribed in further detail below at Section VIII.

V. ISOLATION AND PURIFICATION OF CDK4I

The term “substantially pure” as used herein denotes a protein which issubstantially free of other compounds with which it may normally beassociated in vivo. In the context of the invention, the term refers tohomogenous CDK4I, which homogenicity is determined by reference topurity standards known to those of ordinary skill in the art (e.g.,purity sufficient to allow the N-terminal amino acid sequence of theprotein to be obtained).

Substantially pure CDK4I may be obtained from tissue homogenates(containing “normal” cells; i.e., those cells which contain the CDK4Igene), through microbial expression, by synthesis, or by purificationmeans known to those skilled in the art, such as affinitychromatography. Such techniques may be utilized to obtain biologicallyactive peptide fragments of CDK4I. In this context, “biologically activepeptide fragments” refers to fragments which contain a binding domainfor CDK4.

Determination that a CDK4I fragment contains a CDK4 binding domain maybe made by use of any of several methods known to those skilled in theart, including determination of the binding kinetics and affinity of thefragment for CDK4 as well as inhibition studies using anti-CDK4antibodies (see, e.g., Xiong, et al., Genes Dev., 7:1572-1583, 1993, thedisclosure of which is incorporated herein by this reference toillustrate a standard method for production of anti-CDK4 antibodies;other suitable methods for antibody production which may be adapted toproduce anti-CDK4 antibodies are described infra). Minor modificationsof the primary amino acid sequence of CDK4I (which may be readilyderived from SEQ.ID.Nos. 1-2) may result in variants which havesubstantially equivalent activity as compared to the specific CDK4Iprotein described herein. Such modifications may be deliberate, as bysite-directed mutagenesis, or may be spontaneous. All of the variantsproduced by these modifications are included herein as long asbiological activity present in the original protein still exists. Forpurposes of this disclosure, such variants shall. be generallyconsidered to be “functional variants”. Functional amino acid sequencevariants of CDK4I may fall into one or more of three classes;substitutional, insertional or deletional variants. Such variantsordinarily are prepared by site-specific mutagenesis of nucleotides inthe DNA encoding CDK4I hereby producing DNA encoding the variant, andthereafter expressing the DNA in recombinant cell culture. However,variant CDK4I and CDK4I fragments having up to about 100-150 residuesmay be conveniently prepared by in vitro synthesis.

Amino acid sequence variants are ordinarily characterized by theintended nature of the variation, but such variants also includenaturally occurring allelic or interspecies variation of the CDK4I aminoacid sequence. The variants typically exhibit the same qualitativebiological activity as the naturally-occurring analogue, althoughvariants may also be selected in order to modify the characteristics ofCDK4I as will be more fully described below.

While the site for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be directed at the target codon or regionand the expressed CDK4I variants screened for the optimal combination ofdesired activity. Techniques for making substitution mutations atparticular sites in DNA having a known sequence are well known, forexample M13 primer mutagenesis. Amino acid substitutions are typicallyof single residues; insertions usually will be on the order of aboutfrom 1 to 10 amino acid residues; and deletions will usually range aboutfrom 1 to 30 residues. Deletions or insertions preferably are made inadjacent pairs, i.e., a deletion of 2 residues or insertion of 2residues.

Substitutions, deletions, insertions or any combination thereof may becombined to arrive at a final construct. Obviously, the mutations thatwill be made in the DNA encoding the variant CDK4I must not place thesequence out of reading frame (see, SEQ.ID.Nos: 1-2).

Substitutional variants are those in which at least one residue in SEQID No. 2 has been removed and a different residue inserted in its place.These may be made to eliminate glycosylation sites in the sequence, toalter the pH, to increase the stability of the protein, or to accomplishother desirable modifications in the protein, which modifications willbe apparent to those of ordinary skill in the art. For example,oxidative stability of CDK4I may be achieved by deletion of cysteine orother labile residues. Deletion or substitution of potential proteolysissites can also be accomplished by deleting such residues or substitutinga glutaminyl or histidyl residue.

Insertional amino acid sequence variants of CDK4I are those in which oneor more amino acid residues are introduce into a predetermined site inthe target receptor. Most commonly, insertional variants are fusions ofheterologous proteins or polypeptides to the amino or carboxyl terminusof the protein to be varied. For example, immunogenic CDK4I derivativesmay be made by fusing an immunogenic polypeptide to the target sequenceby cross-linking in vitro or by recombinant cell culture transformedwith DNA encoding the fusion. Such immunogenic polypeptides preferablyare bacterial polypeptides such as trpLE, beta-galactosidase and thelike, together with their immunogenic fragments. CDK4I of the inventionalso includes amino acid sequence mutants, glycosylation variants andcovalent or aggregative conjugates with other chemical moieties.Covalent derivatives of CDK4I may also be prepared by linkage offunctional moieties to groups which are found in the receptor's aminoacid side chains or at the N, or C-termini, by means known in the art.These derivatives may, for example, include aliphatic esters or amidesof the carboxyl terminus or residues containing carboxyl side chains,O-acyl derivatives of hydroxyl group-containing residues, and N-acylderivatives of the amino terminal amino acid or amino-group containingresidues, e.g. lysine or arginine.

Another group of derivatives are covalent conjugates of CDK4I and CDK4Ifragments with other proteins or polypeptides. These derivatives may besynthesized by one of ordinary skill in the art in recombinant cultureas N, or C-terminal fusions or by the use of dysfunctional agents knownper se for use in cross-linking proteins to insoluble matrices throughreactive side-groups.

Covalent or aggregative derivatives will be useful as immunogens,reagents in immunoassay or for affinity purification of CDK4I. Forexample, CDK4I insolubilized by covalent bonding to cyanogen-bromide-activated “SEPHA-ROSE” (agarose tradename) by known methods oradsorbed to polyoefin surfaces may be used in an assay or inpurification of anti-CDK4I antibodies or CDK4I ligand.

With reference to SEQ.ID.Nos: 1-2, CDK4I protein and peptides can beidentified and synthesized by such commonly used methods as t-BOC orFMOC protection of alpha-amino groups. Both methods involve stepwisesyntheses whereby a single amino acid is added at each step startingfrom the C terminus of the peptide (see, Coligan, et al., CurrentProtocols in Immunology, Wiley Interscience, 1991, Unit 9). Peptides ofthe invention can also be synthesized by various well known solid phasepeptide synthesis methods, such as those described by Merrifield (J. Am.Chem. Soc., 85:2149, 1962), and Stewart and Young (Solid Phase PeptidesSynthesis, Freeman, San Francisco, 1969, pp 27-62), using a copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer. Oncompletion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout ¼-1 hours at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution which isthen lyophilized to yield the crude material. This can normally bepurified by such techniques as gel filtration on a “SEPHADEX G-15” or“SEPHAROSE” affinity column. Lyophilization of appropriate fractions ofthe column will yield the homogeneous peptide or peptide derivatives,which can then be characterized by such standard techniques as aminoacid analysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectroscopy, molar rotation,solubility, and quantitated by the solid phase Edman degradation.

Compositions comprising CDK4I may include such substances as thestabilizers and excipients described below, predetermined amounts ofproteins from the cell or organism that served as the source of theCDK4I gene, proteins from other than CDK4I source cells or organisms,and synthetic polypeptides such as poly-L-lysine. Recombinant CDK4Iwhich is expressed in allogeneic hosts will of course be expressedcompletely free of gene source proteins. For example, expression ofhuman CDK4I in Chinese Hamster Ovary (CHO) cells or other nonhumanhigher mammalian cells results in a composition where the receptor isfree of contaminating agents and human proteins.

VI. CDK4I DNA SEQUENCES AND EXPRESSION PRODUCTS

The invention also provides polynucleotides which encode CDK4I. As usedherein, “polynucleotide” refers to a polymer of deoxyribonucleotides orribonucleotides, both single-stranded (including sense and antisensestrands) and double-stranded, in the form of a separate fragment or as acomponent of a larger construct. DNA encoding a peptide of the inventioncan be assembled from cDNA fragments or from oligonucleotides whichprovide a synthetic gene which is capable of being expressed in arecombinant transcriptional unit. Polynucleotide sequences of theinvention include genomic DNA, RNA and cDNA sequences. A polynucleotidesequence can be deduced from the genetic code, however, the degeneracyof the code must be taken into account. Polynucleotides of the inventioninclude sequences which are degenerate as a result of the genetic code.

As described in further detail below, polynucleotide sequences encodingCDK4I can be expressed in either prokaryotes or eukaryotes. Hosts caninclude microbial yeast, insect and mammalian organisms. Methods ofexpressing DNA sequences having eukaryotic or viral sequences inprokaryotes are well known in the art. Biologically functional viraliandplasmid DNA vectors capable of expression and replication in a host areknown in the art. Such vectors (i.e., “recombinant expression vectors”)are used to incorporate DNA sequences of the invention. These sequencesmay also be contained in “host cells”, i.e., transformed cells such asCHO and COS cells (e.g., ATCC Accession No. CRL 1651) for use in geneexpression.

DNA encoding CDK4I is obtained from sources other than humans by a)obtaining a cDNA library from mammalian tissue b) conductinghybridization analysis with labelled DNA encoding human growth hormonereceptor and binding protein or fragments thereof (usually, greater than100 bp) in order to detect clones in the CDNA library containinghomologous sequences, and c) analyzing the clones by restriction enzymeanalysis and nucleic acid sequencing to identify full-length clones. Iffull length clones are not present in the library, then appropriatefragments may be recovered from the various clones and ligated atrestriction sites common to the clones to assemble a full-length clone.

DNA which encodes CDK4I is obtained by chemical synthesis, by screeningreverse transcripts of mRNA from placental cells or cell line cultures,or by screening genomic libraries from any cell. Also included withinthe scope of the invention is nucleic acid which may not encode CDK4Ibut which nonetheless is capable of hybridizing with DNA encoding CDK4Iunder low stringency conditions (e.g. “primers” or “probes”). The probesand primers of the invention will generally be oligonucleotides; i.e.,either a single stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands which may be chemically synthesized. Suchsynthetic oligonucleotides have no 5′ phosphate and thus will not ligateto another oligonucleotide without adding a phosphate with an ATP in thepresence of a kinase. A synthetic oligonucleotide will ligate to afragment that has not been dephosphorylated. Such oligonucleotides maybe detectably labelled with a detectable substance such as a fluorescentgroup, a radioactive atom or a chemiluminescent group by known methodsand used in conventional hybridization assays. Such assays are employedin in vitro diagnosis, such as detection of CDK4I DNA or mRNA in tissuesamples.

In general, prokaryotes are used for cloning of DNA sequences inconstructing CDK4I expressing recombinant expression vectors. Forexample, E. coli K12 strain 294 (ATCC Accession No. 31446) may beparticularly useful. Prokaryotes also are used for expression. Theaforementioned strain, as well as E. coli W3110 (ATTC Accession No.27325), bacilli such as Bacillus subtilus, and other enterobacteriaceaesuch as Salmonella typhimurium or Serratia marcescans, and variouspseudomonas species may also be used for expression.

In general, plasmid vectors which may be used in the invention containpromoters and control sequences which are derived from speciescompatible with the host cell. The vector ordinarily carries areplication site as well as marker sequences which are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322, a plasmid derived from an Ecoli species (Bolivar, et al., Gene, 2:95, 1977). pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR322 plasmid, or othermicrobial plasmid must also contain or be modified to contain promotersand other control elements commonly used in recombinant DNAconstruction.

Promoters suitable for use with prokaryotic hosts illustratively includethe β-lactamase and lactose promoter systems (Chang, et al., Nature,275:615, 1978; and Goeddel, et al., Nature, 281:544, 1979), alkalinephosphatase, the tryptophan (trp) promoter system (Goeddel, NucleicAcids Res., 8:4057,1980) and hybrid promoters such as the taq promoter(de Boer, et al., Proc. Natl. Acad. Sci. USA, 80:21-25, 1983). However,other functional bacterial promoters are suitable. Their nucleotidesequences are generally known in the art, thereby enabling a skilledworker to ligate them to DNA encoding CDK4I (Siebenlist et al., Cell,20:269, 1980) using linkers or adapters to supply any requiredrestriction sites.

In addition to prokaryotes, eukaryotic microbes such as yeast culturesmay also be used. Saccharomyces cerevisiae, or common baker's yeast isthe most commonly used eukaryotic microorganism, although a number ofother strains are commonly available.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman, et al., J. Biol.Chem., 255:2073, 1980) or other glycolytic enzymes (Hess, et al. J. Adv.Enzyme Reg. 7:149, 1968; and Holland, Biochemistry, 17:4900, 1978) suchas enolase, glyceralde-hyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degraded enzymes associated with nitrogen metabolism,metallothionine, glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Yeast enhancers alsoare advantageously used with yeast promoters.

“Control region” refers to specific sequences at the 5′ and 3′ ends ofeukaryotic genes which may be involved in the control of eithertranscription or translation. Virtually all eukaryotic genes have anAT-rich region located approximately 25 to 30 bases upstream from thesite where transcription is initiated. Another sequence found 70 to 80bases upstream from the start of transcription of many genes is a CCAATregion where X may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence which may be the signal for additional ofthe poly A tail to the 3′ end of the transcribed mRNA.

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and later promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers, et al., Nature, 273:113, 1978). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa Hindlil E restriction fragment (Greenaway, et al., Gene, 18:355-360,1982). Promoters from the host cell or related species also are usefulherein.

Transcription of a DNA encoding CDK4I by higher eukaryotes is increasedby inserting an enhancer sequence into the vector. Enhancers arecis-acting elements of DNA, usually about from 10-300 bp, that act on apromoter to increase its transcription. Enhancers are relativelyorientation and position independent having been found 5′ (Laimins, etal., Proc.NatL.Sci.Acad.USA, 78:993, 1981) and 3′ (Lusky, et al., Mol..Cell Bio., 3:1108, 1983) to the transcription unit, and within anintron (Banerji, et al., Cell, 33:729, 1983) as well as within thecoding sequence itself (Osborne, et al., Mol.Cell Bio., 4:1293 1984).Many enhancer sequences are now known from mammalian gene (globin,elastase, albumin, α-feto-protein and insulin). Typically, however, anenhancer from a eukaryotic cell virus will be used. Examples include theSV40 enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription which may affect mRNA expression. Expression vectors mayalso contain a selection gene, also termed a selectable marker. Examplesof suitable selectable markers for mammalian cells which are known inthe art include dihydrofolate reductase (DHFR), thymidine kinase orneomycin. When such selectable markers are successfully transferred intoa mammalian host cell, the transformed mammalian host cell can surviveif placed under selective pressure, (i.e., by being conferred with drugresistance or genes altering the nutrient requirements of the hostcell).

Suitable host cells for transformation with and expression of thevectors of this invention encoding CDK4I in higher eukaryotes include:monkey kidney CV1 line transformed by SV40 (ATCC CRL 1651); humanembryonic kidney line (Graham, F. L., et al., J. Gen Virol., 36:59,1977); baby hamster kidney cells (ATCC CCL 10); chinese hamsterovary-cells-DHFR (Urlaub and Chasin, Proc. Nat'l Sci. Acad. USA,77:4216, 1980); mouse sertoli cells (Mather, J. P., Biol.Reprod.,23:243-251, 1980); monkey kidney cells (ATCC CCL 70); african greenmonkey kidney cells (ATCC CRL-1587); human cervical carcinoma cells(ATCC CCL 2); canine kidney cells (ATCC CCL 34); buffalo rat liver cells(ATCC CRL 1442); human lung cells (ATCC CCL 75); human liver cells (HB8065); mouse mammary tumor (ATCC CCL51); and TRI cells (Mather, et al.,Annals N.Y. Acad. Sci., 383:44-68, 1982).

“Transformation” means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or by chromosomalintegration, such as described in Graham, et al., Virology, 52:456-457,1973. However, other methods for introducing DNA into cells such as bynuclear injection or by protoplast fusion may also be used. Ifprokaryotic cells or cells which contain substantial cell wallconstructions are used, transfection may be achieved by means well knownin the art such as calcium treatment using calcium chloride as describedby Cohen, F. N., et al., (Proc.Nat'l Acad.Sci. USA, 69:2110, 1972). Aparticularly convenient method of transforming host cells is bylipofection using, for example, the liposomal product or DOTMA (atrademarked product of Bethesda Research Labs, Gaithersberg, Md.).

“Transfection” refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisanusing, for example, CaPO₄ or electroporation. Successful transfection isgenerally recognized when any indication of the operation of thetransfected vector occurs within the host cell.

Construction of suitable vectors containing the desired coding andcontrol sequences employ standard ligation techniques. Isolated plasmidsor DNA fragments are cleaved, tailored, and relegated in the formdesired to form the plasmids required.

For example, for analysis to confirm correct sequences in plasmidsconstructed, the ligation mixtures may be used to transform a host celland successful transformants selected by ampicillin or tetracyclineresistance where appropriate. Plasmids from the transformants areprepared, analyzed by restriction and/or sequenced by, for example, themethod of Messing, et al., (Nucleic Acids Res., 9:309, 1981), the methodof Maxam, et al., (Methods in-Enzymology, 65.499, 1980), or othersuitable methods which will be known to those skilled in the art. Sizeseparation of cleaved fragments is performed using conventional gelelectrophoresis as described, for example, by Maniatis, et al.,(Molecular Cloning, pp. 133-134, 1982).

Host cells may be transformed with the expression vectors of thisinvention and cultured in conventional nutrient media modified as isappropriate for inducing promoters, selecting transformants oramplifying genes. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

With reference to SEQ ID NO's: 1-2, production of polynucleotides by theaforementioned techniques is well within the skill of one of ordinaryskill in the art. The invention therefore encompasses CDK4Ipolynucleotides obtained by such techniques.

VII. CDK4I ANTIBODIES

The invention also encompasses polyclonal and monoclonal antibodieswhich specifically bind to CDK4I. Such antibodies can be biologicallyproduced through immunization of a mammal with CDK4I (includingantigenic fragments thereof and fusion proteins), hereafter “immunogenicCDK4I”.

A multiple injection immunization protocol is preferred for use inimmunizing animals with immunogenic CDK4I (see, e.g., Langone, etal.,eds., “Production of Antisera with Small Doses of Immunogen: MultipleIntradermal Injections”, Methods of Enzymology, Acad. Press, 1981). Forexample, a good antibody. response can be obtained in rabbits byintradermal injection of 1 mg of immunogenic CDK4I emulsified inComplete Freund's Adjuvant followed several weeks later by one or moreboosts of the same antigen in incomplete Freund's Adjuvant.

If desired, immunogenic CDK4I molecules may be coupled to a carrierprotein by conjugation using techniques which are well-known in the art.Such commonly used carriers which are chemically coupled to themolecules include keyhole limpet hemocyanin (KLH), thyroglobulin, bovineserum albumin (BSA), and tetanus toxoid. The coupled molecule is thenused to immunize the animal (e.g., a mouse or a rabbit).

Polyclonal antibodies produced by the immunized animals can be furtherpurified, for example, by binding to and elution from a matrix to whichthe peptide to which the antibodies were raised is bound. Those of skillin the art will know of various techniques common in the immunology artsfor purification and/or concentration of polyclonal antibodies, as wellas monoclonal antibodies (see, for example, Coligan, et al., CurrentProtocols in Immunology, Unit 9, (Wiley lnterscience, 1991)).

For their specificity and ease of production monoclonal antibodies willbe preferred for use in detecting CDK4I in analyte samples (e.g., tissuesamples and cell lines). For preparation of monoclonal antibodies,immunization of a mouse or rat is preferred. The term “antibody” as usedin this invention is meant also to include intact molecules as well asfragments thereof, such as for example, Fab and F(ab¹)₂, which arecapable of binding the epitopic determinant. Also, in this context, theterm “mAb's of the invention” refers to monoclonal antibodies withspecificity for CDK4I.

The general method used for production of hybridomas secretingmonoclonal antibodies (“mAb's”) is well known (Kohler and Milstein,Nature, 256:495, 1975). Briefly, as described by Kohler and Milstein,the technique comprised isolation of lymphocytes from regional draininglymph nodes of five separate cancer patients with either melanoma,teratocarcinoma or cancer of the cervix, glioma or lung. The lymphocyteswere obtained from surgical specimens, pooled, and then fused withSHFP-1. Hybridomas were screened for production of antibody which boundto cancer cell lines. An equivalent technique can be used to produce andidentify mAb's with specificity for CDK4I.

Confirmation of CDK4I specificity among mAbs of the invention can beaccomplished using relatively routine screening techniques (such as theenzyme-linked immunosorbent assay, or “ELISA”) to determine theelementary reaction pattern of the mAb of interest.

It is also possible to evaluate an mAb to determine whether is has thesame specificity as mAb of the invention without undue experimentationby determining whether the mAb being tested prevents a mAb of theinvention from binding to CDK4I. If the mAb being tested competes withthe mAb of the invention, as shown by a decrease in binding by the mAbof the invention, then it is likely that the two monoclonal antibodiesbind to the same or a closely related epitope.

Still another way to determine whether a mAb has the specificity of amAb of the invention is to pre-incubate the mAb of the invention with anantigen with which it is normally reactive, and determine if the mAbbeing tested is inhibited in its ability to bind the antigen. If the mAbbeing tested is inhibited then, in all likelihood, it has the same, or aclosely related, epitopic specificity as the mAb of the invention. Asnoted further below, this same general technique may also be used toscreen potential CDK4I ligand.

Methods known in the art also allow antibodies which will specificallybind a preselected ligand to be identified and isolated from antibodyexpression libraries. For example, a method for the identification andisolation of an antibody binding domain which exhibits binding with apeptide of the invention is the bacteriophage y vector system. Thisvector system has been used to express a combinatorial library of Fabfragments from the mouse antibody repertoire in Escherichia coli (Huse,et al., Science, 246:1275-1281, 1989) and from the human antibodyrepertoire Mullinax, et al., (Proc.Nat'lAcad.Sci. USA, 87:8095-8099,1990). As described therein, antibodies which bound a preselected ligandwere identified and isolated from these antibody expression libraries.This methodology can also be applied to hybridoma cell lines expressingmonoclonal antibodies which bind for a preselected ligand.

This invention further provides chimeric antibodies of theCDK4I-specific antibodies described above or biologically activefragments thereof. As used herein, the term “chimeric antibody” refersto an antibody in which the variable regions of antibodies derived fromone species are combined with the constant regions of antibodies derivedfrom a different species or alternatively refers to CDR graftedantibodies. Chimeric antibodies are constructed by recombinant DNAtechnology and are described, for example, in Shaw, et al., J. Immun.,138:4534, 1987, and Sun, LK., et al., Proc.Natl.Acad.Sci. USA,84:214-218, 1987.

In addition, methods of producing chimeric antibody molecules withvarious combinations of “humanized” antibodies are known in the art andinclude combining murine variable regions with human constant regions(Cabily, et al., Proc.Natl.Acad.Sci USA, 81:3273, 1984), or by graftingthe murine-antibody complementary determining regions (CDRs) onto thehuman framework (Riechmann, et al., Nature, 322:323, 1988).

Any of the above described antibodies or biologically active antibodyfragments can be used to generate CDR grafted and chimeric antibodies.“CDR” or “complementarty determining region” or “hypervariable region”are each defined as the amino acid sequences on the light and heavychains of an antibody which form the three-dimensional loop structurethat contributes to the formation of the antigen binding site.

As used herein, the term “CDR grafted” antibody refers to an antibodyhaving an amino acid sequence in which at least parts of one or more CDRsequences in the light and/or variable domain have been replaced byanalogous parts of CDR sequences from an antibody having a differentbinding specificity for a given antigen or receptor.

The terms “light chain variable region” and “heavy chain variableregion” refer to the regions or domains at the N-terminal portion of thelight and heavy chains respectively which have a varied primary aminoacid sequence for each antibody. The variable region of the antibodyconsists of the amino terminal domain of the light and heavy chains asthey fold together to form a three-dimensional binding site for anantibody.

The analogous CDR sequences are said to be “grafted” onto the substrateor recipient antibody. The “donor” antibody is the antibody providingthe CDR sequence, and the antibody receiving the substituted sequencesis the “substrate” antibody. One of skill in the art can readily producethese CDR grafted antibodies using the teachings provided herein incombination with methods well known in the art (see Borrebaeck, AntibodyEngineerng: A Practical Guide (W. H. Freeman and Company, New York,1992)).

Under certain circumstances, monoclonal antibodies of one isotype mightbe more preferable than those of another in terms of their diagnostic ortherapeutic efficacy. For example, from studies on antibody-mediatedcytolysis it is known that unmodified mouse monoclonal antibodies ofisotype gamma-2a and gamma-3 are generally more effective in lysingtarget cells than are antibodies of the gamma-1 isotype. Thisdifferential efficacy is thought to be due to the ability of thegamma-2a and gamma-3 isotypes to more actively participate in thecytolytic destruction of the target cells. Particular isotypes of amonoclonal antibody of different isotype, by using the sib selectiontechnique to isolate class-switch variants (Steplewski, et al., Proc.Nat'l Acad. Sci. USA, 82:8653, 1985; Spira, et al., J. Immunol. Methods,74:307, 1984).

The invention also encompasses cell lines which produce monoclonalantibodies of the invention. The isolation of cell lines producingmonoclonal antibodies of the invention can be accomplished using routinescreening techniques which permit determination of the elementaryreaction pattern of the monoclonal antibody of interest. Thus, if amonoclonal antibody being tested binds and neutralizes the activityassociated with the specific peptide, for example binds CDK4I and blocksCDK4I-mediated biological activity, then the monoclonal antibody beingtested and the monoclonal antibody produced by the cell lines of theinvention are equivalent.

By using the monoclonal antibodies of the invention, it is possible toproduce anti-idiotypic antibodies which can be used to screen monoclonalantibodies to identify whether the antibody has the same bindingspecificity as a monoclonal antibody of the invention. These antibodiescan also be used for immunization purposes (Herlyn, et al., Science,232:100, 1986). Such antiidiotypic antibodies can be produced usingwell-known hybridoma techniques (Kohler and Milstein, Nature, 256:495,1975).

An anti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the monoclonal antibody produced by the cellline of interest. These determinants are located in the hypervariableregion of the antibody. It is this region (paratope) which binds to agiven epitope and, thus, is responsible for the specificity of theantbody. An anti-idiotypic antibody can be prepared by immunizing ananimal with the monoclonal antibody of interest. The immunized animalwill recognize and respond to the idiotypic determinants of theimmunizing antibody and produce an antibody to these idiotypicdeterminants. By using the anti-idiotypic antibodies of the immunizedanimal, which are specific for a monoclonal antibody of the inventionproduced by a cell line which was used to immunize the second animal, itis now possible to identify other clones with the same idiotype as theantibody of the hybridoma used for immunization. Idiotypic identitybetween monoclonal antibodies of two cell lines demonstrates that thetwo monoclonal antibodies are the same with respect to their recognitionof the same epitopic determinant. Thus, by using anti-idiotypicantibodies, it is possible to identify other hybridomas expressingmonoclonal antibodies having the same epitopic specificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody. Thus, theanti-idiotypic monoclonal antibody can be used for immunization, sincethe anti-idiotype monoclonal antibody binding domain effectively acts asan antigen.

VIII. IMMUNOLOGICAL USE OF Anti-CDK4I ANTIBODIES

Once produced as described supra, anti-CDK4I antibodies may be useddiagnostically (e.g., to detect CDK4I in a biological cell sample ormonitor the level of its expression). Preferably, to detect the CDK4Iprotein in premalignant somatic cells, a suitable cell sample will bederived from skin biopsies, sputum specimens, or urinary specimens.Germline cells may be obtained from any convenient source, such as skin,blood, or hair follicles.

CDK4I may be detected and/or bound using anti-CDK4I antibodies in eitherliquid or solid phase immunoassay formats (when bound to a carrier).Examples of well-known carriers for use in solid-phase assay formatsinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation. Examples of types ofimmunoassays which can utilize monoclonal antibodies of the inventionare competitive and non-competitive immunoassays in either a direct orindirect format.

Specific examples of such immunoassays are the radioimmunoassay (RIA)and the sandwich (immunometric) assay. Binding CDK4I using theanti-CDK4I antibodies of the invention can be done utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Those of skill in the art will know, or canreadily discern other immunoassay formats without undue experimentation.

The anti-CDK4I antibodies of the invention may also be detectablylabelled. There are many different labels and methods of labeling knownto those of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include enzymes,radioisotopes, fluorescent compounds, colloidal metals, chemiluminescentcompounds, and bioluminescent compounds. Those of ordinary skill in theart will know of other suitable labels for binding to the anti-CDK4Iantibodies of the invention, or will be able to ascertain such, usingroutine experimentation. Furthermore, the binding of these labels to theanti-CDK4I antibodies of the invention can be done using standardtechniques common to those of ordinary skill in the art. Anotherlabeling technique which may result in greater sensitivity consists ofcoupling the antibodies to low molecular weight haptens. These haptenscan then be specifically detected by means of a second reaction. Forexample, it is common to use haptens for this purpose such as biotin,which reacts with avidin.

The anti-CDK4I antibodies of the invention can also be used for in vivodiagnosis, such as to identify a site of infection or inflammation or tomonitor a particular therapy. In using the anti-CDK4I antibodies of theinvention for the in vivo detection of antigen having a peptide of theinvention, the detectably labeled monoclonal antibody is given in a dosewhich is diagnostically effective. The term “diagnostically effective”means that the amount of detectably labeled anti-CDK4I antibody isadministered in sufficient quantity to enable detection of the sitehaving cells which express CDK4I.

The concentration of detectably labeled anti-CDK4I antibody which isadministered should be sufficient such that the binding to a peptide ofthe invention is detectable compared to the background. Further, it isdesirable that the detectably labeled antibody be rapidly cleared fromthe circulatory system in order to give the best target-to-backgroundsignal ratio.

As a rule, the dosage of detectably labeled anti-CDK4I antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. The dosage of antibody can varyfrom about 0.01 mg/m², to about 500 mg/m², preferably 0.1 mg/m² to about200 mg/m², most preferably about 0.1 mg/m² to about 10 mg/m². Suchdosages may vary, for example, depending on whether multiple injectionsare given, tissue, and other factors known to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agive type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

The anti-CDK4I antibodies of the invention can be used in vitro and invivo to monitor the course of disease therapy. For example, the CDK4Iprotein and peptide fragments of the invention may be useddiagnostically in biological fluids and tissues to monitor the fate ofanti-CDK4I antibodies used therapeutically as described below.

IX. THERAPEUTIC USES OF CDK4I A. Administration of PharmaceuticalCompositions

Because cancers related to deletion of, or polymorphisms in, the genefor CDK4I are causatively related to the loss of, or reduction in, theinhibitory activity of CDK4I, administration of a therapeuticallyeffective amount of CDK4I will delay, if not also prevent, theprogression or onset of such cancers. Also, because many CDK4I genedeletions and polymorphisms are present in cells which are alsogenetically deficient in the ability to produce MTAse, then combinedtherapeutic regimes directed to providing the patient withtherapeutically effective amounts of both CDK4I and MTAse will also beof benefit in delaying, if not also preventing, the progression or onsetof such cancers.

These ends may be achieved through the direct administration ofpurified, synthetic or recombinant CDK4I and, where appropriate, MTAse.Altematively, these ends may be achieved by gene therapy, particularlygene replacement therapy. Means for the production of purified,synthetic or recombinant CDK4I and/or MTAse will be known to, or can bereadily ascertained, by one of ordinary skill in the art in combinationwith the information concerning CDK4I and MTAse provided in thisdisclosure (i.e., at SEQ.ID.Nos 1-5 and 14; see also, FIGS. 2 (a-b)(showing the genomic nucleotide sequence for the CDK4I gene, with exonsunderlined; and, FIG. 10, showing the genomic nucleotide sequence forthe MTAse gene, with the exons underlined).

CDK4I compositions are prepared for administration by mixing CDK4Ihaving the desired degree of purity with physiologically acceptablecarriers. Such carriers will be nontoxic to recipients at the dosagesand concentrations employed. Ordinarily, the preparation of suchcompositions entails combining the particular protein with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Such compositions mayalso be lyophilized and will be pharmaceutically acceptable; i.e.,suitably prepared and approved for use in the desired application.

Given that CDK4I will be absent or of reduced efficacy in malignant orpremalignant cells, cells having that condition will be the preferredtargets for introduction of the CDK4I compositions of the invention.Where, however, the CDK4I abnormalities to be treated are In germline orsomatic cells with no other detectable signs of malignancy,administration may be by any enteral or parenteral route in dosageswhich will be varied by the skilled clinician depending on the patient'spresenting condition and the therapeutic ends to be achieved.

In this regard, “biological activity” of CDK4 refers to the enzymaticreaction stemming from the binding of CDK4 to cyclin D and relatedmolecules during the growth cycle of a human cell. Further, “biologicalactivity” of CDK4I refers to the inhibition of the biological activityof CDK4 stemming from the binding of CDK4 by CDK4I.

Generally, therefore, a “therapeutically effective dosage” of a CDK4Icomposition will be a dosage sufficient to inhibit the biologicalactivity of CDK4 in human cells wherein CDK4I is absent or itsbiological activity is reduced (as a result, for example, of apolymorphism in the gene for CDK4I). To this end, the dosage of CDK4Ican vary from about 0.1 mg/kg to about 300 mg/kg, preferably from about0.2 mg/kg to about 200 mg/kg, in one or more dose administrations daily,for one or several days.

B. Gene Therapy

The present invention identifies mutations in a target sequence of CDK4Ithat are unique to the primary tumor isolated from a subject andmetastatic sites derived from the primary tumor. In the tumor cells, themutated nucleotide sequence is expressed in an altered manner ascompared to expression in a normal cell; therefore, it is possible todesign appropriate therapeutic (as well as diagnostic) techniquesdirected to this specific sequence. Thus, where a cell-proliferativedisorder is associated with the expression of a particular mutated tumorsuppressor gene nucleic acid sequence, a nucleotide sequence thatinterferes with the specific expression of the mutated gene at thetranscriptional or translational level can be used. This approachutilizes, for example, antisense oligonucleotides and/or ribozymes toblock transcription or translation of a specific mutated mRNA, either bymasking that mRNA with an antisense nucleic acid or by cleaving it witha ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). To date, several tumor suppressor genes andoncogenes have been targeted for suppression or down-regulationincluding, but not limited to, p53 (V. S. Prasolov et al., Mol. Biol.(Moscow) 22:1105-1112, 1988); ras (S. K. Anderson et al., Mol. Immunol.26:985-991, 1989; D. Brown et al., Oncogene Res. 4:243-249, 1989); fos(B. Levi et al., Cell. Differ. Dev. 25 (Suppl): 95-102, 1988; D. Mercolaet al., Gene 72:253-265, 1988); and myc (S. 0. Freytag, Mol. Cell. Biol.8:1614-1624, 1988; E. V. Prochownik et al., Mol. Cell. Biol.8:3683-3695, 1988; S. L. Loke et al., Curf. Top. Microbiol. Immunol.141:282-288, 1988).

It is not sufficient in all cases to block production of the targetmutant gene. As described in A. J. Levine, et al., (Biochimica etBiophisica Acta., 1032:119-136, 1990), there are at least five types ofmutations that can contribute to the tumor phenotype. Briefly, Type Imutations are those mutations in genes that result in abnormal proteinproducts, which act in a positive dominant fashion. Examples of suchmutations are those in H-ras and K-ras genes that result in amino acidchanges at positions 12 or 61 in the protein, leading to a protein thatbinds GTP and is constantly signaling for cell growth. Type II mutationsare those that result in overproduction of an oncoprotein, such as thebcr-abI translocation that results in overproduction of a normal mycprotein and an altered abi protein. Type III mutations are loss offunction mutations wherein tumors arise as the result of loss of bothalleles, such as with the retinoblastoma sensitivity gene (Rb) on humanchromosome 13q14 and the Wilm's tumor sensitivity gene localized at11q13. In 75% of colorectal carcinomas, one allele at the p12-p13.3locus of chromosome 17 containing the p53 gene is commonly deleted, andin some cases the other p53 allele which remains in the colorectalcancer cells has been shown to produce a mutant p53 protein thatpresumably contributes to tumorigenesis. Type IV mutations are thosethat result in expression of a protein that does not directly contributeto the growth of cells, but enhances the ability of cancer cells tosurvive. For instance, mutations to the v-erb-A gene results inerythoblasts transformed with the altered gene being kept in thereplication cycle. Type V mutations result from addition of new geneticinformation into tumor cells, commonly by way of a virus. In some casesthe virus integrates its DNA into the cellular genome to produceproteins that bind to cellular negative regulators of growth, such as RBand p53, and thus, in effect, mimic the Type III loss of functionmutation mechanism.

Antisense therapy can be used to block production of mutant proteinsthat act directly to increase the probability of producing neoplasticcells, such as in mechanism Type III, Type IV and Type V mutations thatmimic Type III. Antisense is also therapeutically effective whenmutation is not dominant, for instance when a non-mutant allele remainsthat encodes the proper protein. However, when the mutation is dominant,as in Type I mutations, and in cases wherein either both alleles aredeleted or one is deleted and the other is mutant, as in certain TypeIII mutations, antisense therapy is preferably accompanied byreplacement therapy. In replacement therapy a wild type gene isintroduced into the target cells identified as having a mutant tumorsuppressor gene or protooncogene which results in production of the wildtype protein necessary to forestall development of the neoplasiaassociated with the identified mutant gene(s).

In the case of tumor suppressor genes, it is known that introducing asuppressor gene into cultured cells either causes cell death or causesno discernible changes, however, the cells may no longer be tumorigenicin animals. Thus, in cases where ribozyme and/or antisense therapy isaccompanied by gene replacement therapy, the chances are increased thatthe cell population containing the mutant gene for which the ribozyme orantisense oligonucleotide is specific will no longer contribute todevelopment of neoplasia in the subject being treated.

Synthetic antisense oligonucleotides are generally between 15 and 25bases in length. Assuming random organization of the human genome,statistics suggest that a 17-mer defines a unique sequence in thecellular mRNA in human DNA; a 15-mer defines a unique sequence in thecellular mRNA component. Thus, substantial specificity for a selectedgenetic target is easily obtained using the synthetic oligomers of thisinvention.

In the cell, the antisense nucleic acids hybridize to the correspondingmRNA, forming a double-stranded molecule. The antisense nucleic acids,interfere with the translation of the mRNA, since the cell will nottranslate a mRNA that is double-stranded. Antisense oligomers of about15 nucleotides are preferred, since they are easily synthesized and areless likely to cause problems than larger molecules when introduced intothe target nucleotide mutant producing cell. The use of antisensemethods to inhibit the in vitro translation of genes is well-known inthe art (Marcus-Sakura, Anal.Biochem., 172:289, 1988). Less commonly,antisense molecules which bind directly to the DNA may be used.

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences that encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences associated with productionof a mutated proto oncogene or tumor suppressor gene in an RNA moleculeand cleave it (Cech, J.Amer.Med. Assnp., 260:3030, 1988). A majoradvantage of this approach is that, because they are sequence-specific,only target mRNAs with particular mutant sequences are inactivated.

There are two basic types of ribozymes, namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species, and18-based recognition sequences are preferable to shorter recognitionsequences.

Unmodified oligodeoxyribonucleotides are readily degraded by serum andcellular nucleases. Therefore, as is well known in the art, certainmodifications of the phosphate backbone have conferred nucleaseresistance to antisense DNA. For instance phosphorothioate,methylphosphonate, and α-anomeric sugar-phosphate, backbone-modifiedoligomers have increased resistance to serum and cellular nucleases. Inaddition, methylphosphonates are nonionic and offer increasedlipophilicity to improve uptake through cellular membranes. The use ofmodified oligonucleotides as antisense agents may require slightlylonger or shorter sequences because chemical changes in molecularstructure can affect hybridization (L. A. Chrisey et al., BioPharm,4:36-42, 1991). These backbone-modified oligos bind to a target sequenceand exert their inhibitory effects by blocking the binding of the cell'stranslational machinery to a specific RNA or by inducing ribonuclease Hactivity through the formation of RNA/DNA duplex structures.

The present invention also provides gene therapy for the treatment ofcancer conditions; i.e., cell proliferative disorders that are mediatedby a deletion of, or polymorphism in, the CDK4I gene. Such therapy wouldachieve its effect by introduction of the specific antisensepolynucleotide and/or replacement wild type gene into cells identifiedby the methods of this invention as having the proliferative disordercaused by mutated genes. Whether the cell will require replacement ofthe wild type gene encoding the CDK4I gene as well as antisense therapyto prevent replication of a CDK4I gene bearing a polymorphism must bedetermined on a case by case basis and will depend upon whether themutation has a dominant effect, ie., whether both alleles of the wildtype gene have been destroyed so that total absence of the gene has acell proliferative effect.

Delivery of antisense tumor suppressor polynucleotides specific formutated genes as well as of replacement wild type genes can be achievedusing a recombinant expression vector such as a chimeric virus or acolloidal dispersion system. Preferred for therapeutic delivery ofantisense sequences is the use of liposomes, especially targetedliposomes.

Various viral vectors that can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting one or more sequences ofinterest into the viral vector, along with another gene which encodesthe ligand for a receptor on a specific target cell, for example, thevector is now target specific. Retroviral vectors can be made targetspecific by inserting, for example, a polynucleotide encoding a sugar, aglycolipid, or a protein. Preferred targeting is accomplished by usingan antibody to target the retroviral vector. Those of skill in the artwill know of, or can readily ascertain without undue experimentation,specific polynucleotide sequences which can be inserted into theretroviral genome to allow target specific delivery of the retroviralvector containing the polynucleotides of interest. A separate vector canbe utilized for targeted delivery of a replacement gene to the cell(s),if needed, or the antisense oligonucleotide and the replacement gene canoptionally be delivered via the same vector since the antisenseoligonucleotide is specific only for target gene containing apolymorphism.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence that enables the packaging mechanism to recognize anRNA transcript for encapsidation. Helper cell lines that have deletionsof the packaging signal include, but are not limited to, Ψ2, PA317 andPA12, for example. These cell lines produce empty virions, since nogenome is packaged. If a retroviral vector is introduced into suchhelper cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion can be produced.

Another targeted delivery system for antisense polynucleotides is acolloidal dispersion system. Colloidal dispersidn systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. It has beenshown that large unilamellar vesicles (LUV), which range in size from0.24.0 μm can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,1981). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells. Inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes encoding the antisense polynucleotides at high efficiency whilenot compromising their biological activity; (2) preferential andsubstantial binding to a target cell in comparison to non-target cells;(3) delivery of the aqueous contents of the vesicle to the target cellcytoplasm at high efficiency; and (4) accurate and effective expressionof genetic information (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

Other means for performing gene therapy are known in the art; to wit,Felgner, et al., Science, 247:1465, 1990; Stibling, et al.,Proc.Natl.Sci.Acad. USA, 89:11277-11281, 1992; and, Tang, et al.,Nature, 356:152-154, 1992, (the disclosures of which are incorporatedherein by this reference to illustrate knowledge in the art concerningmethods for performing gene therapy). However, the preferred means forperforming gene therapy of the invention is the administration of suchgenes in “naked”, non-replicating form (i.e., without association with aviral vector, liposome, host cell or equivalent means for expression ofnucleic acids). Further, the preferred routes for administration of suchnaked nucelotides is via injection into skeletal muscle or, mostpreferably, via introduction into tissue which contains a relativelyhigh concentration of antigen presenting cells.

X. CDK4I KITS AND PRODUCTS

For use in the diagnostic research and therapeutic applicationssuggested above, kits are also provided by the invention. In thediagnostic and research applications such kits may include any or all ofthe following: assay reagents, buffers, CDK4I protein and/or fragments,CDK4I recombinant expression vectors, CDK4I oligonucleotides and otherhybridization probes and/or primers, and/or a suitable assay device. Atherapeutic product may include sterile saline or anotherpharmaceutically acceptable emulsion and suspension base for use inreconstituting lyophilized CDK4I or anti-CDK4I suspensions, suitablylabeled and approved containers of CDK4I or anti-CDK4I compositions, andkits containing these products for use in connection with the diagnostickit components as described above.

Such a kit may also comprise a carrier means being compartmentalized toreceive in close confinement one or more container means such as vials,tubes, and the like, each of the container means comprising one of theseparate elements to be used in the method.

For example, one of the container means may comprise a hybridizationprobe that is or can be detectably labelled. A second container maycomprise a cell lysis buffer. The kit may also have containers holdingnucleotide(s) for amplification of the target nucleic acid sequenceand/or a container comprising a reporter-means, such as a biotin-bindingprotein, such as avidin or streptavidin, bound to a reporter molecule,such as an enzymatic, fluorescent, or radionuclide label.

The invention having been fully described, it is further illustrated bythe example below. It will be understood, however, that the invention isnot limited by the examples but is defined by the appended claims.

EXAMPLE I IDENTIFICATION AND CHARACTERIZATION OF THE CDK4I GENE

MTAse cDNA (SEQ ID NO: 14) was isolated and used to probe a humanplacenta lambda phage library. A 2 kilobase Hind III fragment containedthe 3′-end of the MTAse gene by sequence analysis. Chromosome walkingwas performed, starting with the 3′-end of MTAse. Several screeningcycles of the known P1 phage (see, e.g., Pierce, et al., Meth. Enzymol.,216:549-574, 1992) and subsequent lambda phage libraries led to theisolation of clones that encompassed the deleted region in T98G.Restriction fragments of these phage were subcloned, partiallysequenced, and mapped by Southern blotting and poulsed field gelelectrophoresis. FIG. 4 shows the map of human chromosome 9p21 betweenthe MTAP and interferon-β (IFNB) gene loci, focusing on the deletedsegment in the T98G glioma cell line.

The polymerase chain reaction (PCR) was used to determine the frequencyof deletion of several sequence tagged sites (STS) from chromosome 9p in46 different human malignant cell lines (Table 1). Depending on the celltype, either STS 54F, or STS 5Bs was deleted most frequently. Theseresults focused attention on the 50 kilobase region between STS 54F andSTS 5BS.

Eight malignant cell lines with breakpoints between 54F and 5BS werethen analyzed by STS-PCR, with new probes from the intervening region.The deletion maps are shown in FIG. 5. A 19 kilobase lambda phage clone(10B1) identified the most frequently deleted site (see, FIG. 4 (a)).Phage DNA of clone 10B1 was digested with ECORI and subcloned intoECO-RI-cut pBLUESCRIPT 11 SK+ (Stratagene, La Jolla, Calif.). DNAs fromhuman placenta and melanoma cell lines were digested with EcoRI,resolved on a 0.8% agarose gel, and transferred to nylon membranes.Subclones were subjected to automated DNA sequencing. The 4.2Kb subclone10B1-10 contained both the CDK4I and the CDK413′ nucleotide sequences(SEQ ID NO's 1-2 and 4-5) while the CDK4i5′ nucleotide sequence iscontained in a 10A1 subclone.

The sequence of the 10B1-10 subclone from clone 10B1 (FIG. 4 (a))contains a 306 base pair open reading frame. The 3′-end of the codingregion, and the 3′-noncoding region, are located 2.6 kilobases towardthe MTAse gene while the 5′-end of the gene is telomeric to the deletedregion in T98G.

The PCR amplification reactions were carried out in a total volume of 20μl, containing 0.1 μg of DNA, 1×PCR buffer (10 mM) Tris-HC1, pH 8.3, 50mM KC1, 1.5 mM MgC1₂, 0.01% gelatin), 200 μM of each dNTP, 20 ng each ofsense and anti-sense primers, and 0.5 units of Taq DNA polymerase.Thirtyfive cycles were performed (64° C. annealing and 72° C. extension)followed by gel electrophoresis.

CDK4I5′ (SEQ ID NO: 3) is a 126 bp product generated by reversetranscriptase-PCR in cell line H661 (ATCC Accession No. HTB-183) using asense primer (5′-AATTCGGCACGAGGCAGCAT-3′; SEQ ID NO: 24) and ananti-sense primer (5′-TTATTTGAGCTTTGGTTCTG-3′; SEQ ID NO: 25). PCRproducts were subcloned and sequenced. Clone p7-4 (ATCC Accession No.55540) contained the 5′ sequence of the CDK4 inhibitor cDNA. A 139 bpproduct was amplified from clone p7-4 with a sense primer and a newanti-sense primer (5′-TCGGCC-TCCGACCGTAACTA-3′; SEQ ID NO: 26) and usedfor Southern blotting. Blots were hybridized at 65° C. overnigh, washedat 65° C. in 0.1×SSC containing 0.1% SDS, and exposed to X-ray film.

EXAMPLE II DELETION OR POLYMORPHISMS IN THE CDK4I GENE IN CANCER CELLLINES

As shown in FIG. 9, the 46 originally screened malignant cell lines(Table 1) were rescreened with STS-PCR primers, corresponding to theCDK4I′ CDK4I′ and CDK4I3′ CDK413′ exons (SEQ ID NOS:8-11) (SEQ ID NO.'s8-11). Sixty-one percent of melanomas, 87% of gliomas, 45% of non-smallcell lung cancers, and 64% of leukemias have homozygous deletions of theCDK4I CDK4I gene fragment (Table 1).

Melanoma cell line WM266-4 has deleted only the 5′-end of the CDK4inhibitor gene (SEQ ID NO. 3). It was positive for CDK4I′, negative forSTS 5BS, and produced an abnormal 7.0 kilobase band after EcoRIdigestion, electrophoresis and hybridization to a probe from the5′-region of the CDK4 inhibitor gene. On the other hand, melanoma cellline SK-MEL-31 has deleted only the 3′-end of the CDK4I gene (SEQ ID NO.5). The Detroit 462 cell line (a pharyngeal carcinoma) has a 29 kilobasedeletion within the CDK4I gene. It was positive for CDK4I3′, negativefor CDK4I′, but positive for STS-5BS and STS-71F. The latter two STSsare located centromeric to the 5′-end of the CDK4 inhibitor gene.

Reverse transcriptase-polymerase chain reaction (RT-PCR) assays in humancells revealed CDK4 inhibitor gene transcript in normal cells, but notin cancers with established deletions of the CDK4 inhibitor gene (FIG.9).

To perform the assays, mRNA was purified with a “FASTTRACK” Kit(Invitrogen, San Diego, Calif.) and was treated with RNase-free DNase I(Pharmacia) using human placenta DNA as a control to ensure completeDNase I digestion. After first-strand cDNA synthesis with a StratascriptRT-PCR Kit (Stratagene La Jolla, Calif.), cDNA was amplified withCDK4I3′ primers (58° C. annealing and 70° C. extension).

Primers for the control G3PDH gene (5′-TGGTATGGTGGAAGGACT-CATGAC-3′ (SEQID NO:27) and 5′-ATGCCAGTGAGCTTCCCGTTCAGC-3′ (SEQ ID NO:28)) amplified a190 bp product (55° C. annealing and 72° C. extension). RT-PCR's for theCDK4I3′ axon and G3PDH were performed separately and resolved on a 2%agarose gel. The 355 bp RT-PCR product seen in lanes 1, 2 and 4 of FIG.9 derived from cDNA. These results indicate that human cells contain asingle CDK4 inhibitor gene, that is homozygously deleted or rearrangedin the majority of melanomas, gliomas, and leukemias, and in manynon-small cell lung cancers.

EXAMPLE III DETECTION OF A DELETION OF THE CDK4I GENE A. Preparation ofSolid Support Materials for PCR-ELISA

Twenty μl of 2.5 pmol/ μl an aminated oligonucleotides specific for theCDK4I gene in 5 mM 2-[N-morpholino] ehthanesulfonic acid and 1 mM EDTA,pH. 5.5 were placed in each well of a 96 well microtiter plate made ofpolycarbonate (Costar, Cambridge, Mass.). Then 20 μl of 4 mg/ml1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride (EDC, PierceChemical) were added and the plate was incubated at 37° C. for 2 hours.Wells were then washed once with phosphate buffered saline (PBS) andblocked with 1% bovine serum albumin (BSA ) for one hour.

B. Trible Primer PCR Amplification

Using the primers described in SEQ ID NO's: 8-13, genomic DNA obtainedfrom the cell lines identified in Table 1, supra, was amplified asfollows. 0.1 μg of genomic DNA was added to an amplification mixtureconsisting of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, and 0.01%gelatin (PCR buffer), as well as 200 μM of each dNTP, 20 ng each of theprimers, and 0.5 units of Taq DNA polymerase. Thirty cycles wereperformed in a Perkin-Elmer Cetus DNA thermal cycler, each cycleconsisting of denaturation (94° C., 1 minute), annealing (50-55° C., 1minute) and extension (72° C., 1 minute).

C. Detection of Hybridization and Extension of Immobilized Primers

The wells were washed three times with HW buffer (3×SSC, 0.1%N-lauroylsarcosine) and once with blocking buffer (0.5% GENIUS blockingreagant (a trademarked product of Boehringer Mannheim), in 100 mMTris-HCl, pH 7.5, and 800 mM NaCl), and incubated with 80 μl oftetramethylbenzidine and horseradish peroxidase (kikegaard & PerryLaboratories). The reaction was stopped with 80 μl of 1M 0-phosphate atthe appropriate time point. 150 μl each was transferred to anothermicrotiter plate and OD was measured at 450 nm with a microtiter readerfrom Molecular Devices, Menlo Park, Calif.

The results of this assay are summarized in Table 1, supra.

EXAMPLE V DETECTION OF A GERMLINE NONSENSE MUTATION IN DYSPLASTIC NEVUSSYNDROME CELLS

Primers for CDK4I′ (SEQ ID NO's: 8-9) were constructed and the reversetranscriptase polymerase chain reaction (RT-PCR) used to amplify a CDK4Igene transcript in a human lymphoblastoid cell line (GM06921; derivedfrom a human patient with dysplastic nevus syndrome (familial melanoma).Using the technique described by Orita, et al., supra, and/or thetechnique described by Wu, et al., supra, a mutated form of the CDK4Igene transcript was identified in the GM06921 cell line). Sequenceanalysis of the transcript revealed a C to T transition at position 166of the mRNA, which results in a nonsense mutation (s FIG. 6).

EXAMPLE VI DETECTION OF A CDK4I5′ GENE MICRODELETION IN A LEUKEMIA CELLLINE

Primers for CDK4I5′ (SEQ ID NO's: 12-13) were constructed and thereverse transcriptase polymerase chain reaction (RT-PCR) used to amplifya CDK4I gene transcript in a human leukemia cell line, U937 (ATCCAccession No. CRL 1593). Using the technique described by Orita, et al.,supra, and/or the technique described by Wu, et al., supra, a mutatedform of the CDK4I5′ gene transcript was identified in the U937 cell lineand sequenced, showing a microdeletion of 18 base pairs (see, FIG. 7).

SUMMARY OF SEQUENCES

SEQ ID NO: 1 is the nucleotide sequence for the 5′ region of humangenomic CDK4I and includes the exon sequence for the 5′ region of CDK4I.

SEQ ID NO. 2 is the nucleotide sequence for the internal and 3′ regionsof human, genomic CDK4I.

SEQ ID NO's: 3 through 5 are, respectively, the CDKI5′, CDK4I′, andCDK4I3′ exons.

SEQ ID NO's: 6 and 7 are sequences for oligonucleotide primers for theregion between 54F and 5BS of the 9p21 chromosome (i.e., correspondingto clone 10B1).

SEQ ID NO's: 8 through 13 are sequences for oligonucleotide primers forthe CDK4I′, CDK4I3′and CDK4I5′ exons, respectively.

SEQ ID NO: 14 is the full-length genomic nucleotide sequence for MTAse.

28 1146 base pairs nucleic acid single linear DNA (genomic) - 1..1146/note= “5′ region of human genomic CDK4I” exon 390..515 /note= “CDK4I5′exon” 1 TTTGGGGNNA AGTTTGGGAA AANCCAATCC TCCTTCCTTT CCAACNNTGCTTCTGGCGAG 60 GCTCCTTCCC GGCTTGTTCC CCCNGGGGGA AGACCCAACC TGGGCCGACCTTCAGGGTTC 120 CCACATTCCC TAANTGCTCG GAGTTAATAN CACCTCCTCC GAGNACTCGCTCACGNCGTC 180 CCCTTNCCTG GAAAGATACC GCGNTCCCTC NAGAGGATTT GAGGGACAGGGTCGGAGGGG 240 NCTCTTCCGC CAGCACCGGA GGAAGAAAGA GGAGGGGCTG GCTGGTCACCAGAGGGTGGG 300 GCGGACCGCG TGCGCTCGGC GTCTGCGGAG AGGGGGAGAG CAGGCAGCGGGCGGCGGGGA 360 GCAGCATGGA GCCGGCGGCG GGGAGCAGCA TGGAGCCTTC GGCTGACTGGCTGGCCACGG 420 CCGCGGCCCG GGGTCGGGTA GAGGAGGTGC GGGCGCTGCT GGAGGCGGGGGCGCTGCCCA 480 ACGCACCGAA TAGTTACGGT CGGAGGCCGA TCCAGGTGGG TAGAGGGTCTGCAGCGGGAG 540 CAGGGGATGG CGGGCGACTC TGGAGGACGA AGTTTGCAGG GGAATTGGAATCAGGTAGCG 600 CTTCGATTCT CCGGAAAAAG GGGAGGCTTC CTGGGGAGTT TTCAGAAGGGGTTTGTAATC 660 ACAGACCTCC TCCTGGCGAC GCCCTGGGGG CTTGGGAAGC CAAGGAAGAGGAATNAGGAG 720 CCACGCGCGT ACAGATCTCT CGAATGCTGA SAMGATYTTR AGGGSSGRAMATATTTGTAT 780 TCAGATGGAA GTATKCTCTT TATCAGATAC AAAATTTACG AACGTTTGGGATAAAAAGGG 840 AGTCTTAAAG AAATKTAAGA TGTKCTGGGA CTACTTAGCC TCCAATTCACAGATACCTGG 900 ATGGAGCTTA TCTTTCTTAC TAGGAGGGAT TATCAGTGGA AATCTGTGGNGTATGTTGGA 960 ATAAATATCG AATATAAATT TTGATCGAAA TTATTCAGAA GCGGCCGGGCGCGGTGCCTC 1020 ACGCCTTGTA ATCCCTTCAC TTTGGGAGAT CAAGGCGGGG GGGAATCANCTGAGGTCGGG 1080 AGTTCGAGAA CAGCCTGGGC AACAGGTGAA AACCTCGCCT CCTACTAAAAAATACAAAAA 1140 GTAGNC 1146 4286 base pairs nucleic acid single linearDNA (genomic) - 1..4286 /note= “internal and 3′ regions of human genomicCDK4I” exon 192..497 /note= “CDK4I′ exon” exon 3157..3171 /note=“CDK4I3′ exon” 2 GAATTCATTG TGTACTGAAG AATGGATAGA GAACTCAAGA AGGAAATTGGAAACTGGAAG 60 CAAATGTAGG GGTAATTAGA CACCTGGGGC TTGTGTGGGG GTCTGCTTGGCGGTGAGGGG 120 GCTCTACACA AGCTTCCTTT CCGTCATGCC GGCCCCCACC CTGGCTCTGACCATTCTGTT 180 CTCTCTGGCA GGTCATGATG ATGGGCAGCG CCCGAGTGGC GGAGCTGCTGCTGCTCCACG 240 GCGCGGAGCC CAACTGTGCC GACCCCGNCA CTCTCACCCG ACCCGTGCACGACGCTGCCC 300 GGGAGGGCTT CCTGGACACG CTGGTGGTGC TGCACCGGGC CGGGGCGCGGCTGGACGTGC 360 GCGATGCCTG GGGCCGTCTG CCCGTGGACC TGGCTGAGGA GCTGGGCCATCGNGATGTCG 420 CACGGTACCT GCGCGCGGCT GCGGGGGGCA CCAGAGGCAG TAACCATNCCCGNATAGATG 480 CCGCGGAAGG TCCCTCAGGT GAGGACTGAT GATCTNAGAA TTTGNCCCCTGAGAGCTTCC 540 AAAGCTCAGA GNATTCATTT TCCAGCACAG AAAGTNCAGC CCGGGAGANCAGTCTCCGGT 600 CTTGTCTCAG CTCACGCGCC AATCGGTGGG ACGGCCTGAG TCTCCCTATCGCCCTGCCCC 660 GCCAGGGCGG CAAATGGGAA ATAATCCCGA AATGGACTTG CGCACGTGAAAGCCCATTTT 720 GTACATTATA CTTCCCAAAG CATACCACCA CCCAAACACC TACCCTCTGCTAGTTCAAGG 780 CCTAGACTGC GGAGCAATGA AGACTCAAGA GGCTAGAGGT CTAGTGCCCCCTCTTCCTCC 840 AAACTAGGGC CAGTTGCATC CACTTACCAG GTCTGTTTCC TCATTTGCATACCAAGCTGG 900 CTGGACCAAC CTCAGGATTT CCAAACCCAA TTGTGCGTGG CATCATCTGGAGATCTCTCG 960 ATCTCGGCTC TTCTGCACAA CTCAACTAAT CTGAACCTCC TCAGCTAATCTGACCCTCCG 1020 CTTNATGCGG TAGAGTTTAC CAGAGCTGCC CCAGGGGGTT CTGGGGACATCAGGACCAAG 1080 ACTTCGCTGA CCCTGGCAGT CTGTGCACCG GAGTTGGCTC CTTTCCCTCTTAAACTTGTG 1140 CAAGAGATCG CTGAGAGATG AAGGTAGAAT TATGGTCCTC CTTGCCCTNGCCTTTCCTTT 1200 TAGTGATCTC AAAGCATCCT CCCTCCGTCC CCATTCCATG GCCCCAGTTCACTACTCCCA 1260 CAGCTGTCTG GTGAAACTGA CAACATTACT CAATTGTTTC TGGGGGGAGGAACATTTTTT 1320 TTTGAAACAA AATAGATATA TGAAACAGTA CACGGGAATT AACACGATTATTTAAGGTAA 1380 AACATGACCT TGAAGATTAT GAAATCCATC TTATTTTGGC CCAGAACGGGGGCATTGGKC 1440 TCCTTGGCCC ATAGGGGAGC TGGGGAGGAC AGGGTGAAGA GTTAGCTCTAAGCCCTCTNN 1500 TTGGAGATGC TGTAAATACA GAACGCAAAA TCACCTTCGA AGTTAAAGACGCGAAGTTCT 1560 TCTTTACTCG GCCCCTCCTC CCCTCCCCCC CGACAATTCC CTCCAGTTACAGCTAGCATC 1620 CAGGTCCCGG GAGGTGAAGA AGGAGACTTC GGCTCCAGTT ACAGCTAGCATCCGGGTCCC 1680 GATTTAGAAG GAGCTGCCAA TTACAGCGCG GTTCCAGGGC TGAGCAAAAAGCCTGAGGAG 1740 CCAAGTGGGA GAGGGAGTAA AACTACTGAA TTGGGCCACA AGCAAATGAATAAACTGAAC 1800 GACTCTTAAC CAAACCTAAT ATATTTAATC CAAACACACA AGTCTTTCATTTCTTCCCTC 1860 CTCCCTTCCT TCTCTTACTC CCCAACACCC CCTCTTCAAG CACAATTAATTATATGGTTA 1920 GATTCTACTG CGTGATCAGC CCTGTTCTAG GTGGTGGGCA CGCCAAGGTGAATGAGACCA 1980 AACAAGAGTC TTGCCCTCAT GGGGTTTACA TTTGGAGACA GAGTCGATCTGTTGCCCAAC 2040 CTGGAGTGCA GTGGCGCGAT CACAGCTCAC TGCAGCCTCA AACTCCCTGGCTCAAGGGGT 2100 TCTCCCACCT GAGCCTCCCG ACTAGCTGGG ACCACAGGTG CACGCCACGACGCCTGGGTT 2160 TGTTTGTTTG TTTAATAGAG ACGAAGGTCT CACCATGTTA TCTGGGCTCAAGCGATCATC 2220 CCCCCTCCTC CTCCTAAAGT ACTGGGATTA CAGTCCCAAG CTATCTTGCCCGACCTGGGA 2280 AACAGACGTT AAGGAAGATA ACAATCTATT TTCAGAGAGC GAGTTTATAAAACCAATGCA 2340 ATGGGTAAAT ATGAAGTGTG AATAGGAGGA GAAGCTAAAG AGTGGTCGGAGAATCTAATG 2400 CAAGCTACGG GAGAAAGAAA CTCAAGTGCA AATGCTGCCT CAGGAATAAACGTAAAAAGA 2460 GACTTTCAAG TGCAAATGCT CCCTCAGGAA TAAAATAATC TTGAGACTCTCAAGTGTAAA 2520 TGCTGCCTCG GGAGAACCGA ACGGCGAGCT GGAGCCCATA CGCAACGAGATTAGAGAGGA 2580 AGGCAGAAGC CAGAGCACAT GAATAAATGA GCATCCATTT TGTTTCAGAAATGATCGGAA 2640 ACCATTTGTG GGTTTGTAGA AGCAGGCATG CGTAGGGAAG CTACGGGATTCCGCCGAGGA 2700 GCGCCAGAGC CTGAGGCGCC CTTTGGTTAT CGCAAGCTGG CTGGCTCACTCCGCACCAGG 2760 TGCAAAAGAT GCCTGGGGAT GCGGGAAGGG AAAGGCCACA TCTTCACGCCTTCGCGCCTG 2820 GCATTGTGAG CAACCACTGA GACTCATTAT ATAACACTCG TTTTCTTCTTGCAACCCTGC 2880 GGGCCGCGCG GTCGCGCTTT CTCTGCCCTC CGCCGGGTGG ACCTGGAGCGCTTGAGCGGT 2940 CGGCGCGCCT GGAGCAGCCA GGCGGGCAGT GGACTAGCTG CTGGACCAGGGAGGTGTGGG 3000 AGAGCGGTGG CGGCGGGTAC ATGCACGTGA AGCCATTGCG AGAACTTTATCCATAAGTAT 3060 TTCAATGCCG GTAGGGACGG CAAGAGAGGA GGGCGGGATG TTCCACACATCTTTGACCTC 3120 AGGTTTCTAA CGCCTGTTTT CTTTCTGCCC TCTGCAGACA TCCCCGATTGAAAGAACCAG 3180 AGAGGCTCTG AGAAACCTCC GGAAACTTAG ATCATCAGTC ACCGAAGGTCCTACAGGGCC 3240 ACAACTGCCC CCGCCACAAC CCACCCCGCT TTCGTAGTTT TCATTTAGAAAATAGAGCTT 3300 TTAAAAATGT CCTGCCTTTT AACGTAGATA TATGCCTTCC CCCACTACCGTAAATGTCCA 3360 TTTATATCAT TTTTTATATA TTCTTATAAA AATGTAAAAA AGAAAAACACCGCTTCTGCC 3420 TTTTCACTGT GTTGGAGTTT TCTGGAGTGA GCACTCACGC CCTAAGCGCACATTCATGTG 3480 GGCATTTCTT GCGAGCCTCG CAGCCTCCGG AAGCTGTCGA CTTCATGACAAGCATTTTGT 3540 GAACTAGGGA AGCTCAGGGG GGTTACTGGC TTCTCTTGAG TCACACTGCTAGCAAATGGC 3600 AGAACCAAAG CTCAAATAAA AATAAAATAA TTTTCATTCA TTCACTCATTTATTGTCAAC 3660 ATTTATTGAG CACCTATTAC AACAATTTCA TCGCATGGAA GACAGCATCGTTTCTGACAC 3720 TGTTGTTTCA TGTATCTCTT AGAAAAACGC TGCTATTAGA CATCTAACACTATTTATCTT 3780 GAGGTGATAA AATATCAAAA GCCGTGTCTC AAGATCGATG AAATGCGGTTAAAATGATGA 3840 ATAGAAACTC TAGGGGGACC TCATATCGAT AGACTCGAGA CTGGCACATCTGGAGATCCG 3900 TATTTATCCG GCTTCCCCTT CCAGATCACG CGAGGTTTGG GATATTTTGCTCACCAGGCC 3960 TCAGCCAGGT AACTGAATCC AGCCAACCCT GGCCCATAGT CTCGGAATCCGACTCGGCTC 4020 CCAGTCCCCG CCTCGGCGTT CTGAGACCCC CAGGCTGGGT TCCAAGAGGGCTGTGAGGTT 4080 GCGAATGACT GCTGCCAAAC CGGAAGGAAC TCTGCGGTTC TCTGCCACAGTGGGATTGTT 4140 GCAGGCACGC GGCTCAGACT TCACTGAGGT TGGGAGATGC TCCTGTCCACGCTGCCTCAT 4200 CCCGTGCTGG AGCACTGCAC CTCTATTTTT TTTTTTAGGG TACACGCCACATAACATAAA 4260 ACTAAAAATT TTAAAGAGTA GAATTC 4286 126 base pairs nucleicacid single linear DNA (genomic) exon 1..126 /note= “CDK4I5′ exon” 3ATGGAGCCTT CGGCTGACTG GCTGGCCACG GCCGCGGCCC GGGGTCGGGT AGAGGAGGTG 60CGGGCGCTGC TGGAGGCGGG GGCGCTGCCC AACGCACCGA ATAGTTACGG TCGGAGGCCG 120ATCCAG 126 306 base pairs nucleic acid single linear DNA (genomic) exon1..306 /note= “CDK4I′ exon” 4 GTCATGATGA TGGGCAGCGC CCGAGTGGCGGAGCTGCTGC TGCTCCACGG CGCGGAGCCC 60 AACTGTGCCG ACCCCGNCAC TCTCACCCGACCCGTGCACG ACGCTGCCCG GGAGGGCTTC 120 CTGGACACGC TGGTGGTGCT GCACCGGGCCGGGGCGCGGC TGGACGTGCG CGATGCCTGG 180 GGCCGTCTGC CCGTGGACCT GGCTGAGGAGCTGGGCCATC GNGATGTCGC ACGGTACCTG 240 CGCGCGGCTG CGGGGGGCAC CAGAGGCAGTAACCATNCCC GNATAGATGC CGCGGAAGGT 300 CCCTCA 306 15 base pairs nucleicacid single linear DNA (genomic) exon 1..15 /note= “CDK4I3′ exon” 5GACATCCCCG ATTGA 15 20 base pairs nucleic acid single linear DNA - 1..20/note= “CDK4I′ primer” 6 GGAAATTGGA AACTGGAAGC 20 20 base pairs nucleicacid single linear DNA - 1..20 /note= “CDK4I′ primer” 7 CAGGTCATGATGATGGGCAG 20 20 base pairs nucleic acid single linear DNA - 1..20/note= “CDK4I3′ primer” 8 CCCGCTTTCG TAGTTTTCAT 20 20 base pairs nucleicacid single linear DNA - 1..20 /note= “CDK4I3′ primer” 9 CAGAACCAAAGCTCAAATAA 20 20 base pairs nucleic acid single linear DNA - 1..20/note= “5BS primer” 10 GCTTAGTTTT AGAGGGTGAT 20 20 base pairs nucleicacid single linear DNA - 1..20 /note= “5BS primer” 11 CATCACTCATAAGAACTGCT 20 19 base pairs nucleic acid single linear DNA - 1..19/note= “CDK4I5′ primer (sense)” 12 ACCATGGAGC CTTGGCTGA 19 19 base pairsnucleic acid single linear DNA - 1..19 /note= “CDK4I5′ primer(antisense)” 13 CAATAGTTAC GGTCGGAGG 19 2763 base pairs nucleic acidsingle linear DNA (genomic) - 1..2763 /note= “full-lengthmethylthioadenosine phosphorylase (MTAse) genomic nucleotide sequence”exon 254..421 exon 616..720 exon 964..1203 14 TTTATACAGA GCATGACAGTGGGGTCCTCA CTAGGGTCTG TCTGCCACTC TACATATTTG 60 AAACAGGAGT GGCTTCTCAGAATCCAGTGA ACCTAAATTT TAGTTTTAGT TGCTCACTGG 120 ACTGGGTTCT AGGAGACCCCCTGTGTTAGT CTGTGGTCAT TGCTAGSAGA ATCACTTAAT 180 TTTTTCTAGA CTCTAGGAGAAAACAGTTGG TGGTGTACTC ATCACGGGTT AACAATTTCT 240 TCTCTCCTTC CATAGGCATGGAAGGCAGCA CACCATCATG CCTTCAAAGG TCAACTACCA 300 GGCGAACATC TGGGCTTTGAAGGAAGAGGG CTGTACACAT GTCATAGTGA CCACAGCTTG 360 TGGCTCCTTG AGGGAGGAGATTCAGCCCGG CGATATTGTC ATTATTGATC AGTTCATTGA 420 CANNNNNNNN NNNNNNNNNNGAGGTCGACG GTATCGATAA GCTTTGTAAA CAATTGTCTT 480 TAGCTTATCC AGAGGAATTGAGTCTGGAGT AAAGACCCAA ATATTGACCT AGATAAAGTT 540 GACTCACCAG CCCTCGGAGGATGGAAAGAT GGCCTTAAAA TAAAACAAAC AAAAACCTTT 600 TTTGCTTTAT TTTGTAGGACCACTATGAGA CCTCAGTCCT TCTATGATGG AAGTCATTCT 660 TGTGCCAGAG GAGTGTGCCATATTCCAATG GCTGAGCCGT TTTGCCCCAA AACGAGAGAG 720 GTGTGTAGTC TTTCTGGAAGGTGTACCAGA ATAAATCATG TGGGCTTGGG GTGGCATCTG 780 GCATTTGGTT AATTGGCAGACGGAGTGGCC CCATACCCTC ACTCAAGTTT GCTTTGTATT 840 ATGCAAGTTT ATGGAGAGTTATTTCCTGTT GCTAATAATT TNNNNNNNNN NNNNNNNNNN 900 AAGTGCAGCC TTAAGTTGTGCATGTGCTAG TATGTTTTGA AGTTTCTGGT TTTTCTTTTC 960 TAGGTTCTTA TAGAGACTGCTAAGAAGCTA GGACTCCGGT GCCACTCAAA GGGGACAATG 1020 GTCACAATCG AGGGACCTCGTTTTAGCTCC CGGGCAGAAA GCTTCATGTT CCGCACCTGG 1080 GGGGCGGATG TTATCAACATGACCACAGTT CCAGAGGTGG TTCTTGCTAA GGAGGCTGGA 1140 ATTTGTTACG CAAGTATCGCCATGGGCACA GATTATGACT GCTGGAAGGA GCACGAGGAA 1200 GCAGTAGGTG GAATTCTTTTCTAAGCACAT ATAGCATGGG TTTCTGGGTG CCAATAGGGT 1260 GTCTTAACTG TTTGTTTCTATTACGTTAGT TTCAGAAAGT GCCTTTCTAC AAGGTTTTGA 1320 AGTTGTTAAT ATTTTCTGTAGTTCCATTGG AAGGTAAGAA CAAAGATCAA AAGAAAGAAA 1380 GAGACACTTT TACCCAAGGATCAGTAGTGA AAATAGTACA TTGTAGGCAT GTAGATGTGT 1440 TGAGAATCAT ACTAAGACTTGGGCCTTANN NNNNNNNNNN NNNNNNNNNN NNTACCCTAC 1500 ATTGAGGATT CGGTTTCAGCAGATAAATTT GAGGGACACA AACATTTAGG CTGTAGCAAG 1560 GCTGGAGCTC AGAAAAATGTTTTATGACAA GCAGTGGAAT TTTAAGTTCT AGTAACCTCC 1620 AGTGCTATTG TTTCTCTAGGTTTCGGTGGA CCGGGTCTTA AAGACCCTGA AAGAAAACGC 1680 TAATAAAGCC AAAAGCTTACTGCTCACTAC CATACCTCAG ATAGGGTCCA CAGAATGGTC 1740 AGAAACCCTC CATAACCTGAAGGTAAGTGC AGCCATGGAC AATCAGGCAT GTCTGTAGAC 1800 TCTCTATTGT CTTCTTTTCTTACTTGCATT TCACCTTTGG TCCTCATGTA TTTTTTGCCA 1860 GCCTAGATGT TTTCAACAAGTTTTTGTGAC ATCTACTACT ACCATACCAA CCACTTGTGA 1920 AACTGAGTAG TCTTATTTTCTTGGCTGGTA GTGCAGANNN NNNNNNNNNN NNAATAAACA 1980 ATAATCCAGG CTGGGCTGGTATGGCAATAA GTGATTATCA GAACAATGCT CTGAGATAAG 2040 CATTATTAAC CTCACTTTACAGGAAAGGGA GGTGAGGAAC CAAGAGTTTA GAGTACCCGA 2100 AGTTCCACAT CTGGTTAGTGAACTTGAAAA TTTTCTGTAG AATTTATTTA AAGTGTATGT 2160 TTCCTGCGTC CTCACTTTGATCTAGAAAAT CAAAATCTGT TTTTTTTTTT AACAAACATC 2220 TCAGTAATTA CGCCAACATGTGAATATCAC TGCCTCCTTT CTTCCTTTCA GAATATGGCC 2280 CAGTTTTCTG TTTTATTACCAAGACATTAA AGTAGCATGG CTGCCCAGGA GAAAAGAAGA 2340 CATTCTAATT CCAGTCATTTTGGGAATTCC TGCTTAACTT GAAAAAAATA TGGGAAAGAC 2400 ATGCAGCTTT CATGCCCTTGCCTATCAAAG AGTATGTTGT AAGAAAGACA AGACATTGTG 2460 TGTATAGAGA CTCCTCAATGATTTAGACAA CTTCAAAATA CAGAAGAAAA GCAAATGACT 2520 AGTAACATGT GGGAAAAAATATTACATTTT AAGGGGGAAA AAAAACCCCA CCATTCTCTT 2580 CTCCCCCTAT TAAATTTGCAACAATAAAGG GTGGAGGGTA ATCTCTACTT TCCTATACTG 2640 CCAAAGAATG TGAGGAAGAAATGGGACTCT TTGGTTATTT ATTGATGCGA CTGTAAATTG 2700 GTACAGTATT TCTGGAGGGCAATTTGGTAA AATGCATCAA AAGACTTAAA AATACGGACG 2760 TAC 2763 142 base pairsnucleic acid single linear DNA (genomic) 15 CGGCACGAGG CAGCATGGAGCCTTCGGCTG ACTGGCTGGC CACGGCCGCG GCCCGGGGTC 60 GGGTAGAGGA GGTGCGGGCGCTGCTGGAGG CGGTGGCGCT GCCCAACGCA CCGAATAGTT 120 ACGGTCGGAG GCCGATCCAG GT142 142 base pairs nucleic acid single linear DNA (genomic) 16CGGCGGGGAG CAGCATGGAG CCTTCGGCTG ACTGGCTGGC CACGGCCGCG GCCCGGGGTC 60GGGTAGAGGA GGTGCGGGCG CTGCTGGAGG CGGGGGCGCT GCCCAACGCA CCGAATAGTT 120ACGGTCGGAG GCCGATCCAG GT 142 284 base pairs nucleic acid single linearDNA (genomic) 17 CAGGTCATGA TGATGGGCAG CGCCCGAGTG GCGGAGCTGC TGCTGCTCCACGGCGCGGAG 60 CCCAACTGTG NCGACCCCGN CACTCTCACC CGACCCGTGC ACGACGCTGCCCGGGAGGGC 120 TTCCTGGACA CGCTGGTGGT GCTGNANCGG GCCGGGGCGC GGGTGGACGTNCGCGAATNC 180 CTGGGGNCGT CTTTCCGTNG ACCTGGNTTN ANGAGCTTGG NCATCGNGAATNTCGNACGG 240 TACCTNCCCG CNGTTNGGGG GGGACANAGG NAGGAACNAT NCCC 284 283base pairs nucleic acid single linear DNA (genomic) 18 CAGGTCATGATGATGGGCAG CGCCCGAGTG GCGGAGCTGC TGCTGCTCCA CGGCGCGGAG 60 CCCAACTGCGCCGACCCCGC CACTCTCACC CGACCCGTGC ACGACGCTGC CCGGGAGGGC 120 TTCCTGGACACGCTGGTGGT GCTGCACCGG GCCGGGGCGC GGCTGGACGT GCGCGATGCC 180 TGGGGCCGTCTGCCCGTGGA CCTGGCTGAG GAGCTGGGCC ATCGCGATGT CGCACGGTAC 240 CTGCGCGCGGCTGCGGGGGG CACCAGAGGC AGTAACCATG CCC 283 58 base pairs nucleic acidsingle linear DNA (genomic) 19 GATGATGGGC AGCGCCTGAG TGGCGGAGCTGCTGCTGCTC CACGGCGCGG AGCCCAAC 58 58 base pairs nucleic acid singlelinear DNA (genomic) 20 GATGATGGGC AGCGCCCGAG TGGCGGAGCT GCTGCTGCTCCACGGCGCGG AGCCCAAC 58 118 base pairs nucleic acid single linear DNA(genomic) 21 AATTCGGCAC GAGGCAGCAT GGAGCCTTCG GCTGACTGGC TGGCCACGGCCGCGGCCCGG 60 GGTCGGGTAG AGGAGGTGCG GGCGCTGCCC AACGCACCGA ATAGTTACGGTCGGAGGC 118 136 base pairs nucleic acid single linear DNA (genomic) 22AATTCGGCAC GAGGCAGCAT GGAGCCTTCG GCTGACTGGC TGGCCACGGC CGCGGCCCGG 60GGTCGGGTAG AGGAGGTGCG GGCGCTGCTG GAGGCGGTGG CGCTGCCCAA CGCACCGAAT 120AGTTACGGTC GGAGGC 136 1450 base pairs nucleic acid single linear DNA(genomic) - 1..1450 /note= “methylthioadenosine phosphorylase (MTAse)genomic nucleotide sequence” exon 254..421 exon 616..720 exon 964..120323 TTTATACAGA GCATGACAGT GGGGTCCTCA CTAGGGTCTG TCTGCCACTC TACATATTTG 60AAACAGGAGT GGCTTCTCAG AATCCAGTGA ACCTAAATTT TAGTTTTAGT TGCTCACTGG 120ACTGGGTTCT AGGAGACCCC CTGTGTTAGT CTGTGGTCAT TGCTAGSAGA ATCACTTAAT 180TTTTTCTAGA CTCTAGGAGA AAACAGTTGG TGGTGTACTC ATCACGGGTT AACAATTTCT 240TCTCTCCTTC CATAGGCATG GAAGGCAGCA CACCATCATG CCTTCAAAGG TCAACTACCA 300GGCGAACATC TGGGCTTTGA AGGAAGAGGG CTGTACACAT GTCATAGTGA CCACAGCTTG 360TGGCTCCTTG AGGGAGGAGA TTCAGCCCGG CGATATTGTC ATTATTGATC AGTTCATTGA 420CANNNNNNNN NNNNNNNNNN GAGGTCGACG GTATCGATAA GCTTTGTAAA CAATTGTCTT 480TAGCTTATCC AGAGGAATTG AGTCTGGAGT AAAGACCCAA ATATTGACCT AGATAAAGTT 540GACTCACCAG CCCTCGGAGG ATGGAAAGAT GGCCTTAAAA TAAAACAAAC AAAAACCTTT 600TTTGCTTTAT TTTGTAGGAC CACTATGAGA CCTCAGTCCT TCTATGATGG AAGTCATTCT 660TGTGCCAGAG GAGTGTGCCA TATTCCAATG GCTGAGCCGT TTTGCCCCAA AACGAGAGAG 720GTGTGTAGTC TTTCTGGAAG GTGTACCAGA ATAAATCATG TGGGCTTGGG GTGGCATCTG 780GCATTTGGTT AATTGGCAGA CGGAGTGGCC CCATACCCTC ACTCAAGTTT GCTTTGTATT 840ATGCAAGTTT ATGGAGAGTT ATTTCCTGTT GCTAATAATT TNNNNNNNNN NNNNNNNNNN 900AAGTGCAGCC TTAAGTTGTG CATGTGCTAG TATGTTTTGA AGTTTCTGGT TTTTCTTTTC 960TAGGTTCTTA TAGAGACTGC TAAGAAGCTA GGACTCCGGT GCCACTCAAA GGGGACAATG 1020GTCACAATCG AGGGACCTCG TTTTAGCTCC CGGGCAGAAA GCTTCATGTT CCGCACCTGG 1080GGGGCGGATG TTATCAACAT GACCACAGTT CCAGAGGTGG TTCTTGCTAA GGAGGCTGGA 1140ATTTGTTACG CAAGTATCGC CATGGGCACA GATTATGACT GCTGGAAGGA GCACGAGGAA 1200GCAGTAGGTG GAATTCTTTT CTAAGCACAT ATAGCATGGG TTTCTGGGTG CCAATAGGGT 1260GTCTTAACTG TTTGTTTCTA TTACGTTAGT TTCAGAAAGT GCCTTTCTAC AAGGTTTTGA 1320AGTTGTTAAT ATTTTCTGTA GTTCCATTGG AAGGTAAGAA CAAAGATCAA AAGAAAGAAA 1380GAGACACTTT TACCCAAGGA TCAGTAGTGA AAATAGTACA TTGTAGGCAT GTAGATGTGT 1440TGAGAATCAT 1450 20 base pairs nucleic acid single linear DNA - 1..20/note= “sense primer” 24 AATTCGGCAC GAGGCAGCAT 20 20 base pairs nucleicacid single linear DNA - 1..20 /note= “anti-sense primer” 25 TTATTTGAGCTTTGGTTCTG 20 20 base pairs nucleic acid single linear DNA - 1..20/note= “new anti-sense primer” 26 TCGGCCTCCG ACCGTAACTA 20 24 base pairsnucleic acid single linear DNA - 1..24 /note= “primer for control G3PDHgene” 27 TGGTATGGTG GAAGGACTCA TGAC 24 24 base pairs nucleic acid singlelinear DNA - 1..24 /note= “primer for control G3PDH gene” 28 ATGCCAGTGAGCTTCCCGTT CAGC 24

What is claimed is:
 1. An isolated polypeptide comprising a cyclindependent kinase 4 inhibitor (CDK4I) or biologically active fragmentthereof, wherein said CDK4I is encoded by a DNA molecule consisting ofSEQ ID NO: 3, 4, and 5 in order 5′ to 3′.
 2. A pharmaceuticalcomposition comprising a substantially pure polypeptide of claim
 1. 3.The isolated polypeptide of claim 1, wherein the polypeptide comprisesCDK4I.